ognizant Communication Corporation



Gene Expression, Vol. 11, pp. 117-124
1052-2166/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
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Overexpression of CYP2D6 Attenuates the Toxicity of MPP+ in Actively Dividing and Differentiated PC12 Cells

Naomi Matoh,1,2 Seigo Tanaka,1 Masanori Takehashi,1 Marek Banasik,1,3 Todd Stedeford,1,3 Eliezer Masliah,4 Shigehiko Suzuki,2 Yoshihiko Nishimura,2 and Kunihiro Ueda1

1Laboratory of Molecular Clinical Chemistry, Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
2Department of Plastic Surgery, School of Medicine, Kyoto University, Kyoto 606-8507, Japan
3Laboratory of Toxicology and Risk Assessment, Institute of Coal Chemistry, Polish Academy of Sciences, 5 Sowińskiego St., 44-121 Gliwice, Poland
4Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0624

Clonal pheochromocytoma cell lines overexpressing cytochrome P450 2D6 (CYP2D6) were established. CYP2D6 was localized in the endoplasmic reticulum, and its enzymatic activity in the microsomal fraction was confirmed by using high performance liquid chromatography analysis with [guanidine-14C]debrisoquine as a substrate. Overexpression of CYP2D6 protected both actively dividing and differentiated cells against the toxic effects of 1-methyl-4-phenylpyridinium ion at the concentration range of 20-40 mM, as assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The production of reactive oxygen species in the mitochondria was suppressed. The cytotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was unchanged in both actively dividing and differentiated cells overexpressing CYP2D6 versus mock-transfected controls at concentrations up to 500 mM. These results suggest that the lowered enzyme activity of CYP2D6 in individuals termed "poor metabolizers" may represent a risk factor from exposure to select neurotoxicants.

Key words: Debrisoquine hydroxylase; MPTP; Parkinson's disease; Reactive oxygen species

Address correspondence to Seigo Tanaka, M.D., Ph.D., Laboratory of Molecular Clinical Chemistry, Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. Tel: +81-774-38-3225; Fax: +81-774-38-3226; E-mail: seigo@scl.kyoto-u.ac.jp

Gene Expression, Vol. 11, pp. 125-139
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Copyright © 2003 Cognizant Comm. Corp.
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Transcriptomic Classification of Antitumor Agents: Application to the Analysis of the Antitumoral Effect of SR31747A

Jean-Bernard Ferrini,* Omar Jbilo,* Annick Peleraux, Therese Combes, Hubert Vidal, Sylvaine Galiegue, and Pierre Casellas

Immunology-Oncology Department, Sanofi~Synthelabo Recherche, 371 rue Prof. Blayac, F-34184 Montpellier CEDEX 04, France

SR31747A is a sigma ligand that exhibits a potent antitumoral activity on various human tumor cell lines both in vitro and in vivo. To understand its mode of action, we used DNA microarray technology combined with a new bioinformatic approach to identify genes that are modulated by SR31747A in different human breast or prostate cancer cell lines. The SR31747A transcriptional signature was also compared with that of seven different representative anticancer drugs commonly used in the clinic. To this aim, we performed a two-dimensional hierarchical clustering analysis of drugs and genes which showed that 1) standard molecules with similar mechanism of action clustered together and 2) SR31747A does not belong to any previously characterized class of standard anticancer drugs. Moreover, we showed that 3) SR31747A mainly exerted its antiproliferative effect by inhibiting the expression of genes playing a key role in DNA replication and cell cycle progression. Finally, contrasting with other drugs, we obtained evidence that 4) SR31747A strongly inhibited the expression of three key enzymes of the nucleotide synthesis pathway (i.e., dihydrofolate reductase, thymidylate synthase, and thymidine kinase) with the latter shown both at the mRNA and protein levels. These results, obtained through a novel molecular approach to characterize and compare anticancer agents, showed that SR31747A exhibits an original mechanism of action, very likely through unexpected targets whose modulations may account for its antitumoral effect.

Key words: SR31747A; Anticancer drugs; Transcriptional signature; DNA chip; Thymidine kinase

Address correspondence to Pierre Casellas, Sanofi-Synthelabo Recherche, 371 rue du Professeur Joseph Blayac, F-34184 Montpellier cedex 04, France. Tel: (33) 4 67 10 62 90; Fax: (33) 4 67 10 60 00; E-mail: pierre.casellas@sanofi-synthelabo.com

*Both authors contributed equally to this work.

Gene Expression, Vol. 11, pp. 141-147
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Copyright © 2003 Cognizant Comm. Corp.
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Expression of Wnt, Frizzled, sFRP, and DKK Genes in Adult Human Pancreas

R. Scott Heller,1 Tino Klein,1 Zhidong Ling,2 Harry Heimberg,2 Masaru Katoh,3 Ole D. Madsen,1 and Palle Serup1

JDRF Center for Beta Cell Therapy in Europe
1Hagedorn Research Institute, Department of Developmental Biology, DK-2820 Gentofte, Denmark
2Diabetes Research Center, Brussels Free University (VUB), B-1090 Brussels, Belgium
3National Cancer Center Research Institute, Tokyo, Japan

Wnts are important signaling molecules involved in many normal developmental processes in the human body as well as some forms of cancer. Nineteen Wnt genes are found in the human genome, as well as 10 Wnt receptor genes called Frizzled. Two coreceptors called LRP 5 and 6 are critical for Wnt signal transduction. The interaction of the Wnts with the receptors is regulated by two classes of extracellular Wnt or LRP binding proteins called sFRP and Dickkopf (DKK), which modulate Wnt signaling. We have examined the expression of all Wnt family members both in the exocrine portion and in isolated islets of adult human pancreas. RT-PCR analysis of the 1-day cultured exocrine pellet fraction from the islet isolation procedure showed that Wnt 2, 2b, 3, 4, 5a, 5b, 7a, 7b, 14, and 15 were detectable. All 10 Frizzled (Frz) receptors were expressed but only Frizzled 1, 2, 4, 5, and 6 strongly. RT-PCR performed on purified human islets revealed that Wnt 2b, 3, 4, 5a, 7b, 10a, and 14 and Frz 4, 5, and 6 were the most highly expressed. DKK 1, 3, and 4 as well as sFRP 1, 4, and 5 were expressed in the exocrine fraction. sFRP 2 and 3 were detectable but only at low levels. In situ hybridization for Frz 1-7 showed that expression colocalized with the islets of Langerhans. Together the data suggest that active Wnt signaling occurs in adult pancreas and is probably important for physiological functions.

Key words: Human; Wnt; Frizzled; sFRP; In situ hybridization; Gene expression; PCR; Pancreas

Address correspondence to R. Scott Heller, Ph.D., Hagedorn Research Institute, DK 2820 Gentofte, Denmark. Tel: 45 44 43 91 37; Fax: 45 44 43 80 00; E-mail: shll@hagedorn.dk

Gene Expression, Vol. 11, pp. 149-162
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Copyright © 2003 Cognizant Comm. Corp.
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Rapid Hepatocyte Nuclear Translocation of the Forkhead Box M1B (FoxM1B) Transcription Factor Caused a Transient Increase in Size of Regenerating Transgenic Hepatocytes

Xinhe Wang, Dibyendu Bhattacharyya, Margaret B. Dennewitz, Vladimir V. Kalinichenko, Yan Zhou, Rita Lepe, and Robert H. Costa

Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, College of Medicine, 900 South Ashland Ave, Chicago, IL 60607-7170

The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA binding domain. Liver regeneration studies with the -3 kb transthyretin (TTR) promoter-driven FoxM1B transgenic (TG) mice demonstrated that premature hepatocyte nuclear localization of the FoxM1B transgene protein at 16 h following partial hepatectomy (PHx) caused an 8-h acceleration in the onset of hepatocyte DNA replication (S-phase) and mitosis by stimulating earlier expression of cell cycle genes. Whether the FoxM1B transgene protein participates in immediate early events during liver regeneration remains to be determined. Here, we found that the FoxM1B transgene protein translocated to hepatocyte nuclei immediately following PHx, that its nuclear staining persisted for the first 6 h after surgery, and that this translocation was associated with an increase in size of regenerating TG hepatocytes. However, regenerating TTR-FoxM1B liver did not exhibit altered expression of proteins that have been implicated in mediating increased cell size, including serum-and-gucocorticoid-inducible protein kinase (SGK), protein kinase-B/Akt, the tumor suppresser gene PTEN (negative regulator of the PI3K/Akt pathway), c-Myc, or peroxisome proliferation. Moreover, we demonstrated that hepatocyte nuclear translocation of the FoxM1B transgene protein was rapidly induced during the hepatic acute phase response, which occurs during the immediate early stages of liver regeneration. Analysis of cDNA expression arrays identified a number of genes such as immediate early transcription factors (ID-3, Stat3, Nur77), matrix metalloproteinase-9 (MMP-9), and several glutathione S-transferase (GST) isoforms and stress response genes, whose expression is elevated in regenerating TTR-FoxM1B TG livers compared with regenerating wild-type (WT) liver. These liver regeneration studies demonstrate that hepatocyte nuclear translocation of the FoxM1B transgene protein was sustained for the first 6 h after PHx, and was associated with transient hypertrophy of regenerating TG hepatocytes and increased expression of genes that may enhance hepatocyte proliferation.

Key words: Forkhead Box transcription factor; Liver regeneration; Hypertrophy; Fox M1; Immediate early transcription factors; Winged helix domain; Nuclear localization; Liver regeneration

Address correspondence to Dr. Robert H. Costa, Department of Biochemistry and Molecular Genetics (M/C 669), University of Illinois at Chicago, College of Medicine, 900 S. Ashland Ave, Rm. 2220 MBRB, Chicago, IL 60607-7170. Tel: (312) 996-0474; Fax: (312) 355-4010; E-mail: Robcosta@uic.edu

Gene Expression, Vol. 11, pp. 163-179
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Copyright © 2003 Cognizant Comm. Corp.
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Gene Structure, Alternate Splicing, Tissue Distribution, Cellular Localization, and Developmental Expression Pattern of Mouse Deubiquitinating Enzyme Isoforms Usp2-45 and Usp2-69

Natalia Gousseva and Rohan T. Baker

Ubiquitin Laboratory, Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, GPO Box 334, Canberra, ACT 2601, Australia

We have identified a novel mouse gene, Usp2, encoding two ubiquitin-specific proteases (USPs) due to alternate splicing of 5´ exons. Usp2-45 consists of 396 amino acids (45.2 kDa), while Usp2-69 is 619 amino acids (69.5 kDa). Usp2-69 results from the splicing of different combinations of untranslated 5´ exons (1A, 1B, 1C) onto exon 1D and the 40-kDa catalytic core (exons 3-13), while Usp2-45 has exon 2 spliced onto the core. The catalytic core contains the highly conserved motifs of the UBP family of deubiquitinating enzymes. We can find no evidence for a reported 41-kDa isoform (UBP41) in any sequence databases. Usp2-69 is able to form a complex with Usp2-45 and with itself. Antibodies raised against the catalytic core recognized a 69-kDa protein, but did not detect a 45-kDa protein in mouse tissues. Using Northern blot, Western blot, and immunohistochemistry, Usp2 expression was observed in many adult and embryonic tissues including testis, heart, skeletal muscle, diaphragm, brain, kidney, liver, pancreas, lung, and skin. Both Usp2 isoforms were localized to the cytoplasm when overexpressed in COS-7 and NIH3T3 cells. The Usp2 expression pattern indicates that this protein might be involved in specific processes in different types of cells, especially those that are differentiating, and that its function is not restricted to a development of a particular organ.

Key words: Ubiquitin; Ubiquitin-specific protease; UBP41; Differentiation; Apoptosis

Address correspondence to Dr. Rohan T. Baker, Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, GPO Box 334, Canberra, ACT 2601, Australia. Tel: +61 2 6125 3824; Fax: +61 2 6125 4712; E-mail: Rohan.Baker@anu.edu.au

Gene Expression, Vol. 11, pp. 181-197
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Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Gene Expression Profiling of the Hypoxia Signaling Pathway in Hypoxia-Inducible Factor 1a Null Mouse Embryonic Fibroblasts

Ajith Vengellur,1 Barbara G. Woods,1 Heather E. Ryan,2 Randall S. Johnson,2 and John J. Lapres1

1Department of Biochemistry and Molecular Biology and The National Food Safety and Toxicology Center, Michigan State University, East Lansing, MI 48824
2Molecular Biology Section, Division of Biological Sciences, University of California, San Diego, CA 92093

Hypoxia is defined as a deficiency of oxygen reaching the tissues of the body, and it plays a critical role in development and pathological conditions, such as cancer. Once tumors outgrow their blood supply, their central portion becomes hypoxic and the tumor stimulates angiogenesis through the activation of the hypoxia-inducible factors (HIFs). HIFs are transcription factors that are regulated in an oxygen-dependent manner by a group of prolyl hydroxylases (known as PHDs or HPHs). Our understanding of hypoxia signaling is limited by our incomplete knowledge of HIF target genes. cDNA microarrays and a cell line lacking a principal HIF protein, HIF1a, were used to identify a more complete set of hypoxia-regulated genes. The microarrays identified a group of 286 clones that were significantly influenced by hypoxia and 54 of these were coordinately regulated by cobalt chloride. The expression profile of HIF1a -/- cells also identified a group of downregulated genes encoding enzymes involved in protecting cells from oxidative stress, offering an explanation for the increased sensitivity of HIF1a -/- cells to agents that promote this type of response. The microarray studies confirmed the hypoxia-induced expression of the HIF regulating prolyl hydroxylase, PHD2. An analysis of the members of the PHD family revealed that they are differentially regulated by cobalt chloride and hypoxia. These results suggest that HIF1a is the predominant isoform in fibroblasts and that it regulates a wide battery of genes critical for normal cellular function and survival under various stresses.

Key words: Hypoxia; cDNA microarray; HIF1 protein; Genomics; Fibroblast

Address correspondence to John J. LaPres, 402 Biochemistry Building, Michigan State University, East Lansing, MI 48824-1319. Tel: (517) 432-9282; Fax: (517) 353-9334; E-mail: lapres@msu.edu

Gene Expression, Vol. 11, pp. 199-210
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Copyright © 2003 Cognizant Comm. Corp.
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Comprehensive Expression Profiling of Highly Homologous 39 Hox Genes in 26 Different Human Adult Tissues by the Modified Systematic Multiplex RT-PCR Method Reveals Tissue-Specific Expression Pattern That Suggests an Important Role of Chromosomal Structure in the Regulation of Hox Gene Expression in Adult Tissues

Miyako Yamamoto, Daisaku Takai,* Fumiya Yamamoto, and Fumiichiro Yamamoto

Cancer Genetics and Epigenetics Program, Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037

Homeobox genes play a crucial role as molecular address labels in early embryogenesis by conferring cell fate and establishing regional identity in tissues. Homeobox gene expression is not restricted to the early development, but it is also observed in the differentiated cells in adult tissues. To have a better understanding of the functionality of homeobox gene expression in adult tissues in physiological and pathological phenomena, it is important to determine the expression profiles of Hox genes. We established a system to study the expression of 39 human Hox genes by the modified Systematic Multiplex RT-PCR method. Using this system, we have systematically examined their expression in 26 different adult tissues. The results showed tissue-specific differential expression. They also revealed that the posterior tissues generally express more Hox genes than the anterior tissues and that the genes located centrally in the Hox Gene Complexes are expressed in more tissues than the genes located at the 5´ or 3´ end of the complexes. Instead of similar expression patterns among paralogous genes, we found that several neighboring Hox genes on the same chromosomes exhibited similar tissue-specific expression pattern, which may suggest that the regulation of Hox gene expression may be more dependent on chromosomal structure in adult tissues.

Key words: Hox genes; Homeobox genes; Systematic Multiplex RT-PCR; Hierarchical clustering algorithm; Tissue-specific expression; Adult tissues

Address correspondence to Fumiichiro Yamamoto, Ph.D., The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA, 92037. Tel: (858) 646-3116; Fax: (858) 646-3173; E-mail: fyamamoto@burnham.org

*Present address: National Institute of Radiological Sciences, Chiba-shi, Chiba 263-8555, Japan.