|ognizant Communication Corporation|
AN INTERNATIONAL JOURNAL
INCORPORATING ANTI-CANCER DRUG DESIGN
VOLUME 13, NUMBERS 6-10
Oncology Research, Volume 13, pp. 303-307
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Somatic Cell Nuclear Transfer
Ian Wilmut and Lesley Paterson
Roslin Institute, Roslin, Midlothian, EH25 9PS, UK
Embryos produced by nuclear transfer from a patient's somatic cell offer one potential source of embryonic stem cells for treatment of human degenerative diseases. As with all of the approaches to such therapy, this has both strengths and weaknesses. The cells would be histocompatible with the patient's cells, be expected to have a normal life span, and in principle be a source of any other cell type. However, the time taken and the costs involved in the isolation of the appropriate cell population would probably prohibit large-scale application. Clones have been produced from the cells of adults of five species, but similar studies in at least five other species have produced early embryos, but not offspring. A variety of somatic cells have been used as successful nuclear donors. The present procedures have proved to be repeatable, but are very inefficient when typically between 1% and 4% of reconstructed embryos develop to adulthood. The inefficiency is the accumulated effect of failure at all stages of development. There may be differences between species and donor cell type in the precise pattern of loss. This outcome is assumed to reflect the inappropriate expression of a large number of genes whose lethal effect is exerted at different stages. Improvements in the efficiency may depend upon understanding those mechanisms in the early embryo that establish the precise chromatin structure that governs development.
Key words: Somatic cells; Embryo stem cells; Nuclear transfer; Reproductive cloning; Therapeutic cloning
Address correspondence to Ian Wilmut, Roslin Institute, Roslin, Midlothian, EH25 9PS, UK. E-mail: firstname.lastname@example.org
Advanced Cell Technology, One Innovation Drive, Worcester, MA 01605 and Center for Developmental Biology (CDB), 2-2-3 Minatojima-minamimachi, Chuo-ku Kobe, 650-0047, Japan
Mice can now be cloned from cultured and noncultured adult-, fetus-, male-, or female-derived cells. Using the mouse as a model, research is moving towards a comprehensive description of clones generated by somatic cell nuclear transfer. In addition, embryonic stem (ES) cell lines can be generated from adult somatic cells via nuclear transfer (ntES cells). ntES cells contribute to an extensive variety of cell types including neurons in vitro and germ cells in vivo. Recent advances in mouse cloning are reported to illustrate its strengths and promise in the study of mammalian biology and biomedicine.
Key words: Clone; Microinjection; Nuclear transfer (nt); Embryonic stem cell; Reprogramming; Differentiation; Disease model
Address correspondence to Teruhiko Wakayama, Ph.D., Laboratory for Genomic Reprogramming, Center for Developmental Biology (CDB), 2-2-3 Minatojima-minamimachi, Chou-ku, Kobe, 650-0047, Japan. Tel: +81-78-306-1563; Fax: +81-78-306-1566; E-mail: email@example.com
Developmental Origins of Hematopoietic Stem Cells
C. Robin, K. Ottersbach, M. de Bruijn, X. Ma, K. van der Horn, and E. Dzierzak
Department of Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands
Hematopoietic stem cells (HSCs) are at the foundation of the hematopoietic hierarchy and give rise to all blood lineages in the adult organism. A thorough understanding of the molecular, cellular, and developmental biology of HSCs is of fundamental importance, but is also clinically relevant for the advancement of cell replacement therapies and transplantation protocols in blood-related genetic disease and leukemias. While the major anatomical sites of hematopoiesis change during ontogeny, it was long believed that the developmental origin of the adult mammalian hematopoietic system was the yolk sac. However, current studies have shown that the first adult-type HSCs are autonomously generated in the intrabody portion of the mouse embryo, the aorta-gonads-mesonephros (AGM) region, and sublocalize to the dorsal aorta. HSCs are also found in the other large embryonic vessels, the vitelline and umbilical arteries. The intraluminal hematopoietic clusters along these vessels, together with the role of the Runx1 transcription factor in cluster and HSC formation and the HSC/endothelial/mesenchymal Runx1 expression pattern, strongly suggest a vascular endothelial/mesenchymal origin for the first HSCs. Moreover, a transgenic mouse line expressing the GFP marker under the control of the Sca-1 transcriptional regulatory elements (GFP expression marks all HSCs) shows a clear localization of GFP-expressing cells to the endothelial cell layer of the dorsal aorta. Thus, highly enriched GFP-positive AGM HSCs will serve as a basis for the future examination of the cellular and molecular factors involved in the induction and expansion of adult HSCs.
Key words: Hematopoietic stem cells; Aorta-gonads-mesonephros (AGM) region; Endothelium; Runx1; Embryo; Aorta
Address correspondence to Prof. Dr. E. Dzierzak, Department of Cell Biology and Genetics, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Tel: +31-10-408-7169; Fax: +31-10-408-9468; E-mail: firstname.lastname@example.org
RhoGTPases and Their Role in Cancer
Xiaoyu Li and Bing Lim
Cancer Biology, HIM 955 Harvard Institutes of Medicine, Beth Israel Deaconess Medical Center, 77 Avenue Louis Pasteur, Boston, MA 02115
The RhoGTPases constitute a large family of small GTP binding proteins that police many sensitive crossroads in diverse cellular functions. Therefore, it would not be surprising if aberrant RhoGTPase function led to perturbed growth and differentiation, including carcinogenesis and cancer progression. The evidence for a causative connection between RhoGTPases and primary human cancers is still weak but there are increasing findings to support this link. An appreciation of this connection is timely and important to alert readers to the possibilities of new disease mechanisms and new ways to diagnose and treat cancer.
Key words: Cancer and RhoGTPases; Inhibitory RhoGTPases; Gene expression and mutation; GTPase regulators and effectors
Address correspondence to Bing Lim, Cancer Biology, HIM 955 Harvard Institutes of Medicine, Beth Israel Deaconess Medical Center, 77 Avenue Louis Pasteur, Boston, MA 02115. Tel: (617) 667-5256; Fax: (617) 667-3299; E-mail: email@example.com
Alan R. Clarke and Owen J. Sansom
Cardiff School of Biosciences, Cardiff University, Cardiff, CF10 3US, UK
We have used mice deficient in a series of genes with either known or potential tumor suppressive activity to determine the phenotype of loss of function of these genes in the mouse. We have tested a series of endpoints that derive from the hypothesis that loss of an apoptotic program would be predicted to fail to delete cells carrying DNA damage, that this would lead to increased clonogenic survival and thereby to an increased mutation burden and tumor predisposition. For p53 deficiency, we show that loss of the apoptotic program does not translate into an increase in spontaneous mutation rate. However, p53 deficiency can lead to increased clonogenic survival, although this is highly damage-type dependent. Furthermore, p53 deficiency weakly accelerates tumorigenesis associated with inactivation of the adenomatous polyposis coli gene, Apc. We have also analyzed mice mutant for the mismatch repair genes Msh2, Mlh1, and Pms2, describing circumstances in which all of these strains show defective apoptosis, increased clonogenic survival, and increased mutation rate. However, these effects are highly drug-type dependent and the pattern of dependency argues strongly that mutation rate increases as a direct result from loss of the apoptotic program. We have also identified a new role for p53 by intercrossing the p53 and Msh2 mutants, so demonstrating that heterozygosity for p53 accelerates microsatellite instability. Finally, we have analyzed mice mutant for Mbd4 and show that this gene functions in vivo as a tumor suppressor.
Key words: Tumor suppressor activity; Murine small intestine; p53 deficiency; Tumor predisposition
Address correspondence to Alan R. Clarke, Cardiff School of Biosciences, Cardiff University, Cardiff, CF10 3US, UK. Tel: 02920 874609; E-mail: Clarkear@cf.ac.uk
Asterios S. Tsiftsoglou, Ioannis S. Pappas,* and Ioannis S. Vizirianakis
Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece
Hematopoietic stem cells (HSCs) or early progenitors respond to external stimuli in bone marrow and differentiate into cell-restricted lineages of blood cells of limited life span. In leukemias, however, early hematopoietic progenitors self-renew themselves, fail to respond to differentiation signals, and do not undergo programmed cell death (apoptosis). The basic mechanisms of differentiation and apoptosis of leukemia cells have been the long-term objective of our work. By exploiting widely studied murine and human leukemic cell systems as models of hematopoietic cell differentiation, we explored the mechanisms by which pharmaceutical agents initiate differentiation in leukemic systems. In this article, we present the developmental program of MEL cells with emphasis given on the role of commitment to terminal maturation. Commitment is initiated via inducer-receptor-mediated processes and leads to discrete patterns of expression of several genes that contribute to growth arrest at the G1 phase, expression of differentiated phenotype, and differentiation-dependent apoptosis (DDA). Overall, MEL erythroid cell differentiation represents a developmental program with a highly coordinated set of processes that is "triggered" by an inducer and functions via a network of genes and proteins interacting with each other harmonically to give birth to lineage-restricted phenotype.
Key words: Hematopoietic stem cells; Murine erythroleukemia cells; Differentiation; Development program
Address correspondence to Asterios S. Tsiftsoglou, Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece. Tel: +30-310-997631; Fax: +30-310-997645; E-mail: firstname.lastname@example.org
*Present address: Laboratory of Pharmacology, Department of Veterinary Medicine, School of Health Sciences, University of Thessaly, Trikalon 224, 43100 Karditsa, Greece.
John S. Lazo and Peter Wipf
Departments of Pharmacology and Chemistry, University of Pittsburgh, Pittsburgh, PA
Small molecules provide exceptionally useful tools for probing signaling targets relevant for cancer and stem cell differentiation. In contrast to genetic approaches, the application of small molecules generally offers a graded and reversible disruption of a particular pathway. The vast array of theoretical chemical entities that exist in the chemical universe are now becoming available through the production and distribution of chemical libraries generated by both traditional and combinatorial methods, which are suitable for pharmacological use. Convenient and inexpensive cell-free and cell-based assays can be used to identify chemicals that exhibit desirable antisignaling properties. We illustrate a model of how agents targeted against signaling macromolecules involved in cancer, namely dual specificity protein phosphatases, can be identified.
Key words: Small molecules; Protein phosphatases; Cell signaling pathyways
Address correspondence to John S. Lazo, Department of Pharmacology, University of Pittsburgh School of Medicine, E-1346 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail: email@example.com
James E. Trosko and Chia-Cheng Chang
National Food Safety Toxicology Center, Department of Pediatrics and Human Development, Michigan State University, East Lansing, MI 48824
Both reproductive and therapeutic cloning of human stem cells have been made possible with recent technological advances in the isolation of embryonic stem cells and of pluripotent stem cells from adult tissues. We have isolated normal human kidney and human breast epithelial stem cells, as well as having characterized "immortalized" cells from human neuronal and human pancreatic tissue (Trosko et al., Methods 20:245-264, 2000). The isolation was motivated by the stem cell theory of carcinogenesis. Based on the assumption that stem cells would not express connexin genes, nor have functional gap junctional intercellular communication (GJIC), we have demonstrated that the human kidney, breast, neuronal, and pancreatic stem cells can divide either symmetrically or asymmetrically, depending on whether they are grown in microenvironmental conditions that suppress GJIC (the undifferentiated, proliferative state) or induce GJIC (the differentiated state). Normal breast epithelial stem cells appear to be intrinsically "immortal" until induced to express GJIC, at which time, with appropriate substrate and microenvironmental nutrients, they can form three-dimensional "organoids," expressing markers associated with the mature mammary tissue and forming a physical structure very similar to the in vivo budding, ductal structures. The breast stem cells can be prevented from "mortalizing" and can be converted to neoplastic cells, which maintain many phenotypes of the stem cells.
Key words: Adult stem cells; Gap junctions; Connexins; Asymmetric cell division; Stem cell diseases; Evolution of multicellular organisms
Address correspondence to James Trosko, National Food Safety Toxicology Center, Department of Pediatrics and Human Development, Michigan State University, East Lansing, MI 48824. Tel: (517) 353-6346; Fax: (517) 432-6340; E-mail: firstname.lastname@example.org
Li-Qin Liu, Margaret Sposato, Hai-Yan Liu, Tracy Vaudrain, Meng-Jiao Shi, Kristin Rider, Zack Stevens, Jan Visser, Hong-Kui Deng, and Morey Kraus
ViaCell Inc., Worcester, MA 01605
Ex vivo expansion of hematopoietic stem cells (HSCs) has been investigated as a means of enhancing engraftment of transplantation therapies, but current ex vivo expansion methods typically result in a loss of functional stem cell activity. Factors that can selectively expand human HSCs remain elusive. Recently we have isolated three functionally distinct human brain microvascular endothelial cells (HBMVECs) that differ greatly in their ability to support in vitro proliferation of human umbilical cord blood (UBC) CD34+CD38- cells. Using these distinct HBMVEC populations, we have devised a cell-based functional cloning assay to identify a molecule(s) capable of facilitating expansion of HSCs in vitro. A gene encoded for IGFBP-3 (insulin-like growth factor binding protein-3) has been identified. IGFBP-3 mRNA and protein are differentially expressed in distinct HBMVEC populations. In vitro cell proliferation assay and CD34+CD38- immunophenotype analysis showed that the addition of an exogenous IGFBP-3 to cultures of purified CD34+/-CD38-Lin- cells (CD2/CD3/CD14/CD16/CD19/ CD24/CD56/ CD66b/GlyA depleted) enhanced proliferation of primitive hematopoietic cells with CD34+CD38- phenotype, suggesting that IGFBP-3 is capable of expanding primitive human blood cells. These expanded primitive blood cells were illustrated to maintain ability to generate functional progenitors. IGFBP-3 belongs to a family of high-affinity IGFBPs, which binds to IGFs and modulates their actions. IGFBP-3 appears to have intrinsic bioactivity that is independent of IGF binding. We are currently exploring the underlying mechanism by which IGFBP-3 modulates proliferation of primitive hematopoietic cells, and the potential of IGFBP-3 to expand pluripotent human repopulating cells capable of hematopoietic reconstitution of irradiated NOD/SCID recipients.
Key words: Stem cell expansion in vitro; Endothelial cells; Insulin-like growth factor binding protein-3
Address correspondence to Li-Qin Liu, Ph.D., ViaCell Inc., One Innovation Dr./Biotech III, Worcester, MA 01605. Tel: (508) 793-1566; Fax: (508) 793-8966; E-mail: email@example.com
Insights Into the Role of DNA Methylation in Murine Embryonic Stem Cells Using a Modified Tetracycline-Inducible Gene Expression System
Timothy Chevassut1 and Bing Lim2
1John Hughes Bennett Laboratories, Western General Hospital,
Edinburgh, Scotland, EH4 2XU, UK
2Harvard Institutes of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02115
Murine embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mouse blastocyst. Their ability to differentiate into diverse cell types in vitro has served well as a model of early embryonic developmental processes. A powerful strategy for studying the role of key genes during this time is to be able to regulate their expression in a dose-dependent manner. Here, we outline our experience of applying the "tetracycline-inducible system" to study gene function during ES cell differentiation and describe a number of key modifications designed to overcome the central problem of transgene silencing. Finally, we demonstrate how this approach can yield insights into the role of DNA methylation during ES cell differentiation by studying the function of two genes: DNMT3A, involved in the de novo methylation of cytosine bases, and MBD2b, a methyl-dependent co-repressor and putative cytosine demethylase.
Key words: Embryonic stem cells; tet; Methylation; DNMT3A; 5-Azadeoxycytidine
Address correspondence to Bing Lim, Harvard Medical School, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 330 Brookline Avenue, Boston, MA 02215. Tel: (617) 667-5256; Fax: (617) 667-3299; E-mail:firstname.lastname@example.org
Georgia Sotiropoulou,1 Vassilios Rogakos,2 Theodoros Tsetsenis,1 Georgios Pampalakis,1 Nikolaos Zafiropoulos,1 George Simillides,1 Athanassios Yiotakis,2 and Eleftherios P. Diamandis3
1Department of Pharmacy, School of Health Sciences, University
of Patras, Rion, 26500 Patras, Greece
2Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimioupolis Zografou, Greece
3Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
Kallikreins are proteolytic enzymes that constitute a subfamily of serine proteases. Novel kallikrein genes were cloned recently, and it was shown that the human kallikrein family contains 15 genes tandemly aligned on chromosomal locus 19q13.3-q13.4. Based on their altered expression in tumor cells, kallikreins may be involved in the pathogenesis and/or progression of cancer. Evidence is presented that certain kallikreins may be exploited as diagnostic cancer biomarkers. Although the function(s) of novel kallikreins is currently unknown, increasing evidence suggests that kallikreins may participate in regulatory enzymatic cascade(s). Elucidation of the function of novel kallikreins largely depends on the availability of active recombinant proteins. Here, the zymogen for kallikrein 13 was overexpressed in Pichia pastoris and biochemically characterized. It was shown that the kallikrein 13 zymogen displays intrinsic catalytic activity leading to autoactivation. A clipped form of kallikrein 13 was identified, indicating autocatalytic cleavage at the internal bond R114-S115. Mature kallikrein 13 displays trypsin-like activity with restricted specificity on synthetic and protein substrates. Combinatorial P1-Lys libraries of tetrapeptide fluorogenic substrates were synthesized and used for the profiling of the P2 specificity of selected kallikreins. Interestingly, it was shown that human kallikrein 13, similarly to PSA, could specifically cleave human plasminogen to generate angiostatin-like fragments, suggesting that specific kallikreins may have antiangiogenic actions. An understanding of the physiology of human kallikreins is emerging with potential clinical applications.
Key words: Kallikreins; Serine proteases; Human kallikrein 6; Human kallikrein 13; Cancer biomarkers; Angiostatin
Address correspondence to Georgia Sotiropoulou, Department of Pharmacy, University of Patras, Rion, 26500 Patras, Greece. Tel: +302-610-996384; Fax: +302-610-997658; E-mail: G.Sotiropoulou@upatras.gr
Sally Dabelsteen, Jesper T. Troelsen, and Jorgen Olsen
Department of Medical Biochemistry & Genetics, Biochemistry Laboratory C, University of Copenhagen, The Panum Institute, Blegdamsvej 3 DK-2200, Copenhagen N, Denmark
Rapid renewing epithelia such as the epidermis and the intestinal epithelium are maintained by proliferation of undifferentiated stem cells located at specific locations. Recent experiments indicate that stem cells from adult organs might be able to populate tissues other than their tissue of origin. Such findings open the possibility that adult stem cells from different tissues might share common markers. We investigated this by two different approaches. In a first approach we compared the expression profiles from epidermal and intestinal epithelial cells at various stages of differentiation. We found that 108 of 1176 genes analyzed were expressed above background in either keratinocytes or enterocytes and, among these, only 16 genes were expressed in both cell types. Of these 16 genes expressed in both cell types, only five displayed the same shift in expression level during cellular differentiation. Interestingly, all five genes were downregulated during cellular differentiation and represented ubiquitously expressed genes. In the second approach we analyzed the expression of the CC chemokine receptor 6 (CCR6), which we have recently identified as an early differentiation marker of epidermal cells, in the intestine. This analysis demonstrates that the CCR6 protein is found in enterocytes at later stages of differentiation.
Key words: Keratinocytes; Enterocytes; Expression profiles; CC chemokine receptor 6 (CCR6)
Address correspondence to Jorgen Olsen, Department of Medical Biochemistry & Genetics, Biochemistry Laboratory C, University of Copenhagen, The Panum Institute, Bldg. 6.4., Blegdamsvej 3, DK-2200 N, Copenhagen, Denmark. Tel: +45 35327637; Fax: +45 35367980; E-mail: email@example.com
Abdellah Mansouri, Qingxiu Zhang, Lon D. Ridgway, Ling Tian, and Francois-Xavier Claret
Department of Molecular Therapeutics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030
Chemoresistance is a major impediment to the successful treatment of cancer. It involves various mechanisms, including defects in the apoptosis program that is induced by anticancer drugs. To further explore the mechanisms underlying the development of chemoresistance in ovarian carcinoma after cisplatin (CDDP) treatment, we compared the effect of CDDP on expression of X-linked inhibitor of apoptosis protein (XIAP), a direct inhibitor of caspase-3, -7, and -9, Fas, Fas-ligand (Fas-L), and pro- and antiapoptotic proteins in a CDDP-sensitive human ovarian carcinoma cell line (2008) and its CDDP-resistant subclone (2008C13). In this article, we show that cisplatin treatment led to a differential expression of distinct apoptotic targets in the CDDP-sensitive cell line (2008) and its CDDP-resistant subclone (2008C13). The acquisition of cisplatin resistance was associated with the ability of the treated cells to enhanced expression of XIAP, whereas the death inducer Fas-L was abrogated in 2008C13 following treatment with CDDP. However, the CDDP-sensitive cells failed to activate XIAP but increased Fas-L expression, indicating that distinct regulatory mechanisms are operative. These findings suggest that the expression of XIAP and downregulation of Fas-L are linked to chemoresistance in ovarian carcinoma cells and may represent one of the potential antiapoptotic mechanisms involved during this process.
Key words: Cisplatin; Apoptosis; Chemoresistance; XIAP; Fas-L; Ovarian carcinoma
Address correspondence to Dr. Francois-Xavier Claret, Department of Molecular Therapeutics, Box 317, The University of Texas M.D. Anderson Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel: (713) 792-6306; Fax: (713) 792-4005; E-mail: firstname.lastname@example.org
Andrei L. Gartel, Claudine Feliciano, and Angela L. Tyner
Department of Molecular Genetics, University of Illinois at Chicago, 900 S. Ashland Ave. Chicago, IL 60607
The tumor suppressor p53 is the most frequently mutated gene in human tumors. In response to DNA damage, aberrant growth signals, or chemotherapeutic drugs, p53 is stabilized and induces apoptosis and/or cell cycle arrest. While the mechanisms of p53-dependent apoptosis are not well understood, p53-dependent cycle arrest is primary mediated by the CDK inhibitor p21. p53 is a transcriptional activator and it is not surprising that a majority of p53 mutations occur in the core DNA binding domain and affect DNA binding and transactivation of p53 targets in tumors. We used the capability of p53 to activate transcription for developing a new assay that permits rapid determination of the status of p53 in cancer cell lines of different origin. Our strategy involved using a retrovirus containing a p53-regulated lacZ reporter gene that was introduced into colon and breast tumor cell lines to determine p53 status. Simple staining for b-galactosidase allowed us to confirm that the colon cancer cell lines LIM1215 and HCT116, as well as the breast cancer cell line MCF7, have wild-type p53, and the colon cancer cell line Caco-2 as well as breast cancer cell lines MDA-MB-435 and MDA-MB-231 have mutant p53. This method may be applied to novel cell lines of any origin with unknown status of p53.
Key words: p53; Mutations; Transcription; b-Galactosidase; Retrovirus
Address correspondence to Andrei L. Gartel, Department of Molecular Genetics, 900 S. Ashland Ave., University of Illinois at Chicago, Chicago, IL 60607. Tel: (312) 996-1855; Fax: (312) 413-0353; E-mail: email@example.com
Michael S. Serfas and Angela L. Tyner*
University of Illinois College of Medicine, Department of Molecular Genetics, Chicago, IL 60607
The tyrosine kinases Brk/PTK6/Sik, Srm, Frk/Rak/Gtk/Iyk/Bsk, and Src42A/Dsrc41 have a low degree of sequence homology to other known kinases, including one another. We show here that the exon structure of these kinases, which we will call the Brk family, is highly conserved and distinct from each of the major families of intracellular kinases containing SH3, SH2, and tyrosine kinase domains, including c-Src and Fyn. Brk/Sik and Srm are 1.1 kb apart on human chromosome 20q13.3 and likely are the result of duplication in cis. Several Brk family kinases have an inhibitory effect on Ras pathway signaling from receptor tyrosine kinases. Members of this family can act either in the membrane or at the nucleus, and may change localization patterns depending on external stimuli. Brk has been shown to phosphorylate two proteins in vivo: Sam68, a substrate for Src in mitosis that can substitute for Rev in nuclear export of RNAs; and BKS, a novel adaptor molecule. Brk also functions as a rapid downstream signaling intermediate following calcium-induced differentiation in keratinocytes. It is possible that Brk family kinases may share common functions and interaction partners, which remain for the most part unexamined.
Keywords: BRK; Sik; Srm; Frk; Rak; Src42A
Address correspondence to Angela L. Tyner, University of Illinois College of Medicine, Department of Molecular Genetics, M/C 669, 900 S. Ashland Ave., Chicago, IL 60607. Tel: (312) 996-7964; Fax: (312) 413-0353; E-mail: firstname.lastname@example.org
*Present address: Michael S. Serfas, University of Wisconsin-Madison, Room 205 Bock, 1525 Linden Drive, Madison, WI 53706.
Structural Genomics of Lipid Signaling Domains
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0580
Signaling domains have been identified by the analysis of data derived from biochemical studies, molecular cloning, or genetic studies. With the availability of genomic information from many organisms and the improved sensitivity in homology detection techniques, many new domains are being identified. In an attempt to understand biochemical and biological function of these domains, we have started a small-scale structural genomics, or structural biology with genomic approach. Two examples from our recent work are steroidogenic acute regulatory protein (StAR)-related lipid-transfer (START) domain and inositol polyphosphate 5-phosphatase catalytic (IPP5C) domain. Crystal structure of human MLN64-START domain revealed a hollowed-out protein containing a hydrophobic tunnel just large enough to bind one molecule of cholesterol and completely exclude it from solvent. This structure suggests that the START domain is a classical type of lipid transporter. On the contrary, the function of IPP5C domain has been extensively studied for a long time, but its catalytic mechanism, positional selectivity, and diverse substrate specificity remained mysterious due to the unavailability of three-dimensional structure. With the structural genomic approach, the first structure of IPP5C domain was solved from a S. pombe protein that is now known as SPsynaptojanin and the structure gave us answers to some of these questions.
Key words: Signaling domains; Structural genomics; START domain; IPP5C domain
Address correspondence to Yosuke Tsujishita at his present address: 250 Melvin Calvin Lab, Department of Chemistry, University of California, Berkeley, CA 94720. Tel. (510) 486-4910; Fax: (510) 486-6240; E-mail: YTsujishita@lbl.gov
Hao Xu and M. Raafat El-Gewely
Department of Biotechnology, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway
Mutations in p53 gene could lead to loss of function, negative complementation, or to gain-of-oncogenic functions, thus leading to the increase of tumorigenicity and invasiveness of cancer. This study focused on cancer-related p53 mutants, including A138T, C141Y, R158L, G245C, and R248Q. Using a modified differential display technique, response profiles of plasmid-expressed wild-type as well as mutated p53, in comparison to p53-null cells, are being established. These profiles can help our understanding of p53 involvement in cancer, leading to accurate diagnosis, treatment, and prognostic analysis by: 1) comprehensive knowledge of p53 network gene expression profiles; 2) finding the most significant gene expression profiles of p53 mutants; 3) revealing genes that only respond to p53 mutants (gain of function). Our results showed significant differences in the expression patterns among p53-null, wild-type p53, and p53 mutants A138T, C141Y, R158L, G245C, and R248Q samples. We also report here the first found p53 mutant-triggered alternative splicing.
Key words: p53; Mutation; Differential display; Expression profile; Alternative splicing; Transient expression
Address correspondence to M. Raafat El-Gewely, Department of Biotechnology, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway. Tel: 47-776-44654; Fax: 47-776-45350; E-mail: email@example.com
Marc Zeicher,1,3 Pierre Spegelaere,1 Marie Horth,1 David Gancberg,1 Abdelatif Karim,2 and Francis Dupont2
1Department of Molecular Biology, Université Libre
de Bruxelles, Brussels, Belgium
2Molecular Oncology Unit, Institut Bordet, Université Libre de Bruxelles, Brussels, Belgium
3Medicine Evaluation Unit, Ministry of Health, Brussels, Belgium
We replaced capsid genes by reporter genes and assessed expression in different types of human cancer cells and their normal counterparts, either at the level of whole cell population (CAT ELISA) or at the single cell level [FACS analysis of green fluorescent protein (GFP)]. CAT expression was substantial (up to 10,000 times background) in all infected tumor cells, despite variations according to the cell types. In contrast, no gene expression was detected in similarly infected normal cells (with the exception of an expression slightly above background in fibroblasts). FACS analysis of GFP expression revealed that most tumor cells expressed high level of GFP while no GFP-positive normal cells could be detected with the exception of very few (less than 0.1%) human fibroblast cells expressing high level of GFP. We also replace capsid genes by genes coding for the costimulatory molecules B7-1 and B7-2 and show that, upon infection with B7 recombinant virions, only tumor cells display the costimulatory molecules and their immunogenicity was increased without any effect on normal cells. Using a recombinant minute virus of mice (MVM) containing the herpes simplex thymidine kinase gene, we could get efficient killing of most tumor cell types in the presence of ganciclovir, without affecting normal proliferating cells. The prospects and limitations of these different strategies are discussed.
Key words: Autonomous parvovirus; Oncoselective; Vector; Cancer therapy; Costimulation; Thymidine kinase
Address correspondence to Marc Zeicher, Medicine Evaluation Unit, Ministry of Health, Boulevard Bischoffsheim 33, Brussels 1000, Belgium. Tel: 32-22275623; Fax: 32-22210327; E-mail: firstname.lastname@example.org
Nicolas Olivier Fortunel, Jacques Alexandre Hatzfeld, Marie-Noëlle Monier, and Antoinette Hatzfeld
Laboratoire de Biologie des Cellules Souches Humaines, CNRS-UPR 9045, Institut André Lwoff, 94800 Villejuif, France
A major obstacle to the use of adult somatic stem cells for cell therapy is our current inability to fully exploit stem cell self-renewal properties. The challenge is to obtain defined culture systems where cycling of primitive stem/progenitor cells is stimulated, while their differentiation and senescence are prevented. The cytokine transforming growth factor-b1 (TGF-b1) appears as a potential regulator of hematopoietic stem/progenitor cell self-renewal, as it participates in the control of cell proliferation, survival/apoptosis, and cell immaturity/differentiation. TGF-b1 acts via a complex regulatory network involving intracellular signaling molecules and cell surface receptors. According to the High Proliferative Potential-Quiescent (HPP-Q) cell working model that we introduced previously, TGF-b1 maintains primitive hematopoietic stem/progenitor cells in a quiescent or slow cycling state, in part by downmodulating the cell surface expression of mitogenic cytokine receptors, thus preventing cells from responding rapidly to a mitogenic signal. We have established that this modulation concerns the tyrosine kinase receptors KIT and FLT3, and the IL-6 receptor (IL-6R), three important cytokine receptors controlling early human hematopoietic stem/progenitor cell development. In this article, we show a similar modulation by TGF-b1 of a fourth receptor: the TPO receptor (MPL). As a consequence, TGF-b1 decreased the cell cycle entry of stem/progenitor cells stimulated by the respective ligands of these receptors, the cytokines SF, FL, IL-6, and TPO, whereas neutralization of TGF-b1 increased the cell responsiveness to these mitogenic cytokines. Other aspects of the function of TGF-b1 in the regulation of early hematopoiesis (i.e., the control of stem/progenitor cell survival and immaturity) are reviewed in the discussion.
Key words: Transforming growth factor-b; Hematopoietic stem cells; Progenitor cells; Self-renewal
Address correspondence to Jacques Alexandre Hatzfeld, Laboratoire
de Biologie des Cellules Souches Humaines, CNRS-UPR 9045, Institut André
Lwoff, 94800 Villejuif, France. Tel: 33 1 49 58 33 15; Fax: 33 1 49 58
33 15; E-mail: email@example.com