|ognizant Communication Corporation|
AN INTERNATIONAL JOURNAL
INCORPORATING ANTI-CANCER DRUG DESIGN
VOLUME 16, NUMBER 12
Oncology Research, Vol. 16, pp. 549-556
0965-0407/08 $90.00 + .00
Copyright © 2008 Cognizant Comm. Corp.
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Cellular Studies of MrDb (DDX18)
Sandy Dubaele and Patrick Chène
Druggability-Enzymology-Profiling Unit, Oncology Research, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
Helicases are present in viruses, prokaryotes, and eukaryotes, and several of them have been linked to human diseases. Here we study the role of one putative DEAD-box RNA helicase, MrDb (DDX18), in tumor cells. We show that MrDb is a nucleolar protein ubiquitously expressed in tumor cells and that it is more abundantly expressed in proliferating cells. Inhibition of MrDb with dominant negative mutant or a shRNA reduces tumor cell proliferation without inducing a cell cycle arrest or apoptosis. These findings suggest that MrDb is important for cell proliferation and that its inhibition could prevent tumor cell proliferation.
Key words: Helicase; Target validation; MrDb; DDX18; DEAD box
Address correspondence to Dr. Patrick Chène, WKL125 442, CH-4002 Basel, Switzerland. Fax: +41616963835; E-mail: firstname.lastname@example.org
Antisense Experiments Demonstrate an Exon 4 Minus Splice Variant mRNA as the Basis for Expression of tNOX, a Cancer-Specific Cell Surface Protein
Xiaoyu Tang,1 D. James Morré,2 and Dorothy M. Morré1
1Department of Foods and Nutrition,
Purdue University, West Lafayette, IN 47907, USA
2Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA
A novel hydroquinone and NADH oxidase with protein disulfide-thiol interchange activity (designated ENOX2 or tNOX), associated exclusively with the outer leaflet of the plasma membrane at the surface of cancer cells and in sera of cancer patients, is absent from the surface of noncancer cells and from sera from healthy individuals. Transfection of HeLa (human cervical carcinoma) cells with antisense oligonucleotides and measurement of mRNA levels by real-time quantitative PCR and growth and drug response by in vitro cytotoxicity assays were combined to demonstrate encoding of a cancer-specific and growth-related cell surface protein, tNOX, via an exon 4 minus splice variant. tNOX mRNA levels of HeLa cells were determined following transfection with antisense relative to control cells transfected with Lipofectamine using the cycle threshold method normalized for GAPDH mRNA. Antisense to tNOX exon 4 mRNA blocked generation of full-length tNOX mRNA but not of exon 4 minus mRNA. Antisense to exon 5 mRNA inhibited the production of exon 4 minus mRNA and full-length tNOX mRNA. Scrambled antisense to exon 5 mRNA was without effect. Antisense to exon 5 mRNA decreased the amount of tNOX protein on the surface of cancer cells. As a control, antisense-mediated downregulation of exon 5 minus mRNA of tNOX also was demonstrated as detected using exon 4/exon 6 primers. Exon 5 antisense blocked the cell surface expression of tNOX whereas exon 4 antisense was without effect. In contrast to nontransfected HeLa cells, cells transfected with exon 5 antisense were not inhibited by the green tea catechin, (-)-epigallocatechin-3-gallate. A relationship of tNOX to unregulated growth of cancer cells was provided by data where growth of HeLa cells was inhibited by transfection with the exon 5 antisense oligonucleotides. Growth inhibition was followed by apoptosis in greater than 70% of the transfected cells.
Key words: Alternative splicing; ENOX2; tNOX; Antisense; NADH oxidase; Cancer
Address correspondence to D. James Morre´, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 136 Hansen Life Sciences Research Building, 201 S. University Street, West Lafayette, IN 47907-2064, USA. Tel: 765-494-1388; Fax: 765-494-4007; E-mail: email@example.com
Metabolites of the Radiosensitizer Nicotinamide Are Unlikely to Contribute to the Degree of Emesis Observed With the Parent Drug
Mark W. Ruddock and David G. Hirst
School of Biomedical Sciences, University of Ulster, Newtownabbey, County Antrim, Northern Ireland, BT37 OQB, UK
Nicotinamide is a potent radiosensitizer currently employed in the treatment of cancer of the bladder, head, and neck. Unfortunately, nicotinamide is also a potent emetic at the concentrations required for radiosensitization. Previously, we have demonstrated that nicotinamide-induced emesis is the direct result of decreased spontaneous peristaltic activity in the ileum. However, the effect of nicotinamide's metabolites on peristaltic activity have not been investigated, although several studies have unsuccessfully attempted to correlate the degree of emesis with the levels of the metabolites in plasma. Isolated rat ileum rings and rat tail arteries were perfused with oxygenated Krebs solution in an organ bath. Nicotinamide, 1-methynicotinamide, or N-oxide nicotinamide were introduced to the perfusate and changes in amplitude of spontaneous peristaltic activity or phenylephrine-induced vasoconstriction recorded. Nicotinamide inhibited peristalsis in the ileum and agonist-induced vasoconstriction in the rat tail arteries, as previously observed. However, the primary metabolites of nicotinamide were without effect. Gut smooth muscle and rat tail artery are sensitive to the relaxant effects of nicotinamide. The primary metabolites of nicotinamide are not vasoactive and do not blunt either spontaneous or agonist-induced contraction and are thus unlikely to contribute to the degree of emesis observed following nicotinamide administration.
Key words: Nicotinamide; N-oxide nicotinamide; 1-Methylnicotinamide; Rat tail artery; Vascular smooth muscle; Ileum; Phenylephrine
Address correspondence to Mark Ruddock at his current address: Molecular Biology, Randox Laboratories Ltd, 55 Diamond Road, Crumlin, Northern Ireland, UK. Tel: 02894 451061; E-mail: firstname.lastname@example.org
Adenosine Modulates Cell Growth in the Human Breast Cancer Cells Via Adenosine Receptors
Mojtaba Panjehpour1 and Fatemeh Karami-Tehrani2
1Department of Clinical Biochemistry,
School of Pharmacy & Isfahan Pharmaceutical Sciences Research Center,
Isfahan University of Medical Sciences, Isfahan, Iran
2Department of Clinical Biochemistry, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran
Adenosine modulates the proliferation, survival, and apoptosis of many different cell types. The present study was performed to investigate the role of adenosine receptors in the human breast cancer cell lines MCF-7 and MDA-MB468. The biological effects of adenosine on the cells were analyzed by adenylyl cyclase and cell viability assay as well as RT-PCR of adenosine receptors. RT-PCR results show the expression of the transcript of all adenosine receptors in both cell lines. By using adenosine and selective adenosine receptor agonists or antagonists, we found that A3 stimulation reduced cell viability, which was abolished by pretreatment with A3 receptor antagonist. Moreover, we demonstrated that adenosine (natural agonist) triggers a cytotoxic signal via A3 receptor activation that was not seen for other subclasses of adenosine receptors. Intracellular cAMP concentration was changed significantly only for A3 and A2B receptor-selective agonists, which indicates the functional form of these receptors on the cell surface. In conclusion, our findings revealed the role of adenosine receptors in breast cancer cell lines on growth modulation role of A3 and functional form of A2B, although its involvement in cell growth modulation was not seen. Theses findings as well as data by others may provide a possible application of adenosine receptor agonists/antagonists in breast malignancies.
Key words: Adenosine; Adenosine receptor(s); Human breast cancer cell lines; RT-PCR; cAMP
Address correspondence to Mojtaba Panjehpour, Assistant Professor of Biochemistry, Department of Clinical Biochemistry, School of Pharmacy & Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Post Box 81745-359, Isfahan, Iran. E-mail: email@example.com
Association of CYP1A1 Polymorphisms With Breast Cancer in North Indian Women
Neetu Singh,1 Amit Kumar Mitra,1 Vivek Kumar Garg,2 Amit Agarwal,3 Mandira Sharma,2 Rashmi Chaturvedi,2 and Srikanta Kumar Rath1
1Genotoxicity Laboratory, Toxicology
Division, Central Drug Research Institute, Lucknow 226 001, Uttar Pradesh,
2Lucknow Cancer Institute, Lucknow 226 001, Uttar Pradesh, India
3Department of Endocrine Surgery, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGI), Lucknow 226 014, Uttar Pradesh, India
Cytochrome P-450 (CYP) 1A1 is a candidate gene for low penetrance breast cancer (BC) susceptibility. Evidences demonstrate that ethnic differences in BC incidence may be partly due to genetic factors, including polymorphisms in the genes. In the present case control study four CYP1A1 gene polymorphisms, m1 (T6235C), m2 (A4889G), m3 (T5639C), and m4 (C4887A) were studied for their association with BC conjointly with the known risk factors such as age, menopausal status, diet, and life style. Polymorphisms of CYP1A1 gene were detected by PCR-RFLP method. The homozygous mutant (G/G) of m2 polymorphism was significantly associated with BC. Consequently, association of both m2 heterozygous mutant genotype (A/G) and combined group [homozygous (G/G) plus heterozygous (A/G) mutant genotype] showed association with postmenopausal women. Incidences of BC were also found to be independent of clinicopathological factors except heterozygous mutant genotype (A/G) m2 showed association with dietary factors and high grade tumors while homozygous mutant (G/G) m2 showed association with ER/PR-positive BC cases. Wildtype m3 was observed in all the subjects in cases as well as in controls. No significant association was observed between m1 and m3 polymorphisms and BC risk in all the subjects as well as when stratified into pre- and postmenopausal subjects. This indicates that out of m1 and m2 polymorphisms that have been reported in Asians, only m2 is associated with North Indians.
Key words: Breast cancer; CYP1A1 polymorphism
Address correspondence to Neetu Singh, Ph.D., Central Drug Research Institute, M.G. Marg, Lucknow -226 001, Uttar Pradesh, India. Tel: 91-0522-2412611-18, ext. 4316; Fax: 91-0522-2223405; E-mail: firstname.lastname@example.org