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Role of Carbohydrate-mediated Cell Recognitionand Adhesion in the Progression and
Metastasis of Malignant Cells
Program and Abstracts
November 6. 1998International Conference Hall
Aichi Cancer CenterNagoya Japan
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Aichi Cancer Center International Symposium V
Role of carbohydrate-mediated cell recognitionand adhesion in the progression and
metastasis of malignant cells
Program and Abstracts
Committee of the Aichi Cancer Center International SymposiumChairperson: Makoto Ogawa
Kozo MoritaSuketami TominagaIsao SatohHideo ItoYoshio YamamotoTsuyoshi KitohShigeo NakamuraToshitada TakahashiReiji Kannagi
Organizing Committee of the 5th SymposiumChairperson: Reiji Kannagi
Hayao NakanishiNozomu HiraiwaAkiko KanamoriHiroshi KumimotoShigeo NakamuraTakashi Nonaka
November 6, 1998Alchi Cancer Center, Nagoya Japan
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Program of Symposium
9:20-9:30 Opening RemarksMakoto Ogawa (Aichi Cancer Center)
9:30-10:40 Carbohydrate Determinants in Cancer, Part 1(Chairperson: Dr. T Muramatsu)
9:30-10:10Functional organization of glycosphingolipids at the tumor cell surfacemembraneDr. Sen-itiroh Hakomori (Pacific Northwest Research Institute, SeattleWashington, U.S.A.)
10:10-10:40Roles of glycosphingolipids in the regulation of cell proliferation anddifferentiationDr. Koichi Furukawa (Department of Biochemistry II, Nagoya University,School of Medicine)
10:40-11:50 Carbohydrate Determinants in Cancer, Part 2(Chairperson: Dr. K. Furukawa)
10:40-11:20Selectins, mucins and carcinoma metastasis.Dr. Ajit Varki, (Glycobiology Program, Cancer Center, Division of Cellularand Molecular Medicine, University of California, San Diego, USA)
11:20-11:50Modification of cell surface sialic acid.Akiko Kanamori (Aichi Cancer Center)
11:50-13:00 Lunch
13:00-14:10 Carbohydrate Modification of Signaling Molecules(Chairperson: Dr. M. Ishii)
13:00-13:40Dynamic O-glycosylation of the estrogen receptor, tumor suppressorsand oncogene proteins: A potential role for O-GlcNAc in oncogenesis.Dr. Gerald W. Hart (Department of Biological Chemistry, Johns HopkinsUniversity, Schools of Medicine, USA)
13:40-14:10Circulating autoantibody to estrogen receptor in estrogen-dependent cancerDr. Masaru Ishii (Saitama Cancer Center)
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14:10-16:10 Implication of Selectins in Malignant Disorders(Chairperson: Dr. R. Kannagi)
14:10-14:50Molecular basis of leukocyte recognition of endothelial selectinsDr. Geoffrey S. Kansas (Dept. Microbiology-Immunology, NorthwesternUniv., Medical School, Chicago, Illinois)
14:50-15:20L-selectin ligand: Molecular cloning and characterization of an N-acety1glucosamine 6-O-sulfotransferase involved in synthesis of 6-sulfosialyl Lewis XDr. Takashi Muramatsu (Department of Biochemistry, Nagoya University,School of Medicine)
15:20-15:40 Coffee Break
15:40-16:10Transcriptional regulation of selectin ligand expression in ATL cellsDr. Nozomu Hiraiwa (Expenimental Pathology, Aichi Cancer Center)
16:10-17:40 Detection of Cancer Cells in Circulation, Lymph Nodes andPeritoneal Cavity (Chairperson: Dr. H. Ohkura)
16:10-16:40Clinical value of genetic detection for rare cancer cells in circulation andlymph nodesDr. Nakamori (The 2nd Department of Surgery, Osaka University School ofMedicine)
16:40-17:10Molecular diagnostic detection of free cancer cells in the peritoneal cavityof patients with gastrointestinal and gynecological malignanciesDr. Hayao Nakanishi (Pathology, Aichi Cancer Center)
17:10-17:40Tissue-specific mRNA in cancer detected in peripheral blood by RT-PCR asa novel tumor markerDr. Kazuhiko Uchida (Department of Biochemistry and MolecularOncology, Institute of Basic Medical Sciences, University of Tsukuba,Tsukuba 305, Japan)
17:40-17:45 Concluding RemarksSuketami Tominaga (Aichi Cancer Center)
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Functional Organization of Glycosphingolipids at theTumor Cell Surface Membrane
Sen-itiroh HakomoriDivision of Biomembrane Research, Pacific Northwest Research Institute,Seattle Washington, U.S.A.
Many tumor cells are characterized by accumulation of a large quantityof specific glycosphingolipids (GSLs), many of which have been characterizedas tumor-associated antigens (1). Typical examples are GM3 in mouse B16melanoma, LacCer in mouse mammary carcinoma, GD3 and GD2 in humanmelanoma and neuroblastoma, Gb3 in Burkitt lymphoma, and fucosyl-GM1 insmall cell lung carcinoma. Such tumor-associated GSLs form large clusters atthe plasma membrane bilayer, closely associated with signal transducermolecules such as c-Src, other Src family kinases, Rho, Ras, and FAK (2).Stimulation of GSLs by soluble antibodies, adhesion to antibody-coated plate, oradhesion to plate coated with complementary GSL induces phosphorylation ofc-Src, other Src family kinases, and FAK, and enhanced binding of Rho and Rasto GTP. As an example, stimulation of GM3 in B16 melanoma cells by celladhesion to plate coated with Gg3 or with anti-GM3 antibody DH2 activates c-Src within 3 min, FAK within 15 min, and enhances GTP binding of Rho andRas within 15 min (3). GM3 was found to mediate adhesion of B16 cells tononactivated endothelial cells through GM3-Gg3 or GM3-LacCer interaction,and thereby activate cell motility. GM3-dependent B16 cell adhesion is thereforeconsidered to initiate the metastatic process (4). The activation of varioustransducer molecules by such adhesion is important in explaining the functionalrole of tumor-associated GSLs. A question remains whether GSL-enrichedmicrodomain is independent from well-described caveolae, morphologicallydistinct invaginations of plasma membrane characterized by the presence of thedistinctive scaffold protein caveolin. Numerous studies in recent years indicatethat caveolae play an essential role in endocytosis and signal transduction (e.g 5,6).
In B16 melanoma cells, a membrane subfraction representing GSL-enriched microdomain was separated completely from caveolin-containingsubfraction (caveolae), by immunoseparation technique using anti-GM3 DH2.GM3, c-Src, and Rho were completely absent from caveolin-containingsubfraction, which was characterized by an abundance of cholesterol, but a
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surprisingly very small quantity of sphingomyelin. Ras was also present incaveolin-containing subfraction. Phosphorylation of c-Src and two othermembrane-bound kinases (Mr 45,000 and 29,000) was induced by stimulation ofGM3 in isolated GSL-enriched subfraction, indicating that GSLs andtransducers are functionally associated within the membrane (7); this assemblyis hereby termed "glycosignaling domain". A similar situation was found forLacCer in mammary carcinoma F28-7. Stimulation of LacCer in LacCer/ c-Src-enriched membrane subfraction enhances phosphorylation of a few bandscontaining tyrosine phosphate (Handa K, Hakomori S, unpubl. data).
Such GSL-enriched microdomains are also present in various normalcells and tissues, although the quantities of GSLs and associated signaltransducers are much less than in tumor cells. GD3 is closely associated withLyn (p53/56) in normal rat brain, as evidenced by co-immunoprecipitation ofthese two components with anti-GD3 ganglioside. Stimulation of GD3 by anti-GD3 R24 m primary cerebellar culture results in a 3-fold increase of Lynactivity within 1 min. This indicates a functional connection between GD3 andLyn in normal cells (8). A major question remains whether there is anyqualitative difference in function of GSL-enriched microdomain between normaland tumor cells.
References
1. Hakomori S (1989). Adv Cancer Res 52: 257-331.2. Yamamura S, Handa K, Hakomori S (1997). Biochem Biophys Res
Commun 236: 218-222.3. Iwabuchi K, Yamamura S, Prinetti A, Handa K, Hakomori S (1998). J Biol
Chem 273: 9130-38.4. Kojima N, Shiota M, Sadahira Y, Handa K, Hakomori S (1992). J Biol
Chem 267: 17264-70.5. Brown DA, London E (1997). Biochem Biophys Res Commun 240: 1-7.6. Okamoto T, Schlegel A, Scherer PE, Lisanti MP (1998). J Biol Chem 273:
5419-22.7. Iwabuchi K, Handa K, Hakomori S, submitted for publication.8. Kasahara K, Watanabe Y, Yamamoto T, Sanai Y (1997). J Biol Chem 272:
29947-53.
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Sen-itiroh Hakomori
Born February 13, 1929, Sendai, Japan. Graduated from Tohoku Univ. School ofMedicine, received D. Med. Sci. 1956. Graduate studies followed by facultyappointment, Dept. of Biochemistry, Tohoku Univ. School of Medicine, 1953-59.Professor of Biochemistry, Tohoku College of Pharmaceutical Sciences, 1959-63. Came to U.S. in 1963. Studies at Massachusetts General Hospital andHarvard Medical School, 1963-66. Visiting Professor, Dept. of Biochemistry,Brandeis Univ., 1966-68. Associate Professor, 1968-1971. Professor ofPathobiology and Microbiology, Univ. of Washington, Seattle, 1971-present.Member and Program Head, Biochemical Oncology, Fred Hutchinson CancerResearch Center, 1975-87. Scientific Director, The Biomembrane Institute,1987-96. Head, Division of Biomembrane Research, Pacific NorthwestResearch Institute, 1996-present.
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Roles of Glycosphingolipids in the Regulation of CellProliferation and Differentiation
Koichi Furukawa, M.D.Department of Biochemistry II, Nagoya University School of Medicine65 Tsurumai, Showa-ku, Nagoya 466-0065 Japan.
Glycosphingolipids have been thought to play roles as recognitionmolecules in cell-cell or cell-extracellular matrix interaction, and as modulatorsof cellular signals for proliferation and/or differentiation. Since a number ofglycosyltransferase genes were isolated, it has become possible to investigatethe roles of carbohydrates in vivo by manipulating those genes and modulatingthe profiles of carbohydrate components expressed on the cell surface.
We have, so far, isolated four glycosyltransferase genes responsible forthe synthesis of relatively simple glycosphingolipids. Many trials ofmodification of glycolipids by the introduction of those genes into cultured celllines revealed that they might have more influence on the cell proliferation/differentiation than expected before, and some times determine the cell fates.
When rat pheochromocytoma PC12 was transfected with GD3 synthasegene, it showed enhanced proliferation and inability to respond to nerve growthfactor (NGF) in terms of neurite extension. Continuous phosphorylation of NGFreceptor (Trk) and activation of MAP kinases were observed in thesetransfectants. On the other hand, when GM1/GD1b/GA1 synthase gene wasintroduced, PC12 cells also showed no response to NGF stimulation, while theirgrowth rate did not alter. However, MAP kinases were activated after NGFtreatment in these cells with different time course from that of the parent cell,i.e., with later peak at ca 1 hour after stimulation and an elongation of activationfor more than 2 hours. All these data indicated that changes of glycosphingolipidexpression might give rise to a drastic modulation of cellular signalingmolecules and of the quality of individual signals, leading the cells to theunexpected destiny.
How can glycosphingolipids interact with and modify the structures andfunctions of growth factor receptors? Is the interaction direct or indirect? We donot know the answer yet. In GD3 synthase gene transfectants of PC12, dimerformation of TrkA without NGF stimulation was observed, suggesting that thealteration of glycolipids induced conformational changes of TrkA resulting inthe autonomous dimenization. This fact may partially explain the continuous
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activation of TrkA and MAP kinases, but not the enhanced proliferation. Resultsof inhibition of the proliferation of GD3 synthase gene transfectants withvarious kinase inhibitors revealed that the enhanced proliferation was actuallymediated via Trk/Ras/MEK/ MAPK pathway which usually mediates a signalfor cell differentiation.
Thus, Trk/Ras/MEK/MAPK pathway appears to transduce bothdifferentiation signals and proliferation signals. What are essential differencesbetween NGF-treated PC12 and GD3 synthase gene-introduced PC12? Levels ofactivation of Ras/MEK/MAPK pathway, kinetic pattern of the activation, andpresence/absence of activation of another signal pathway in these two situationsare now being investigated. Also, differences in transcription factors actuallyexpressed in those cells seems critical to finally determine the cell fates, andremain to be analyzed.
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Koichi Furukawa, M.D.
Professor, Department of Biochemistry IINagoya University School of Medicine65 Tsurumail, Showa-ku,Nagoya 466-0065, Japan
1975 Nagoya University School of Medicine, Nagoya (MD)1975-1976 Internship, Nagoya Higashi Citizen Hospital1976-1979 Resident in Internal Medicine,
Nagoya National Hospital1980 Internist, Chunoh Hospital1980-1984 Clinical Resident in Internal Medicine,
Nagoya University School of Medicine1984-1988 Research Associate, Memorial
Sloan-Kettering Cancer Center1988-1990 Assistant Professor, Department of Oncology,
Nagasaki University School of Medicine1990-1992 Lecturer1992-1997 Associate Professor1997-present Professor, Department of Biochemistry II,
Nagoya University School of Medicine
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Selectins, Mucins and Carcinoma Metastasis
Ajit VarkiGlycobiology Program and Cancer Center, University of California, San Diego,La Jolla, CA 92093-0687, USA
Metastasis is a complex multi-step cascade involving many factors thatfavor survival of the "fittest" tumor cells, which have to enter the bloodstream,evade various host defenses, interact with the endothelium of distant organs, andfinally invade and successfully colonize tissues. Substantial evidence fromearlier investigations indicates that circulating platelets and leukocytes interactwith tumor cells when they enter the blood-stream, creating tumor microembolithat are thought to facilitate the process of metastasis. It is also presumed thatthe circulating tumor cells must interact with endothelial cells when they arriveat their final destination.
A consistent and prominent feature of malignant progression is thealteration of cell-surface carbohydrates. In particular, expression of sialyl LewisX has been associated with a poor prognosis in most human carcinomas thathave been studied to date. Expression of sialomucins by carcinomas is alsoassociated with poor prognosis. In keeping with this, inhibition of O-glycosylation has been shown to diminish metastases in some experimentalsystems. However, the mechanisms by which the expression of sialyl Lewis Xand sialomucins facilitates tumor progression is unknown. Given the well-known role of vascular sialomucins bearing sialyl Lewis X as ligands for theselectins, it is reasonable to speculate that the selectins are involved in tumorprogression. Indeed, several investigators have provided indirect evidence thatthis may be the case.
Using recombinant probes, we have found that clinical specimens ofprimary colon carcinomas do express variable levels of binding for each of thethree selectins (P-,E- & L- selectins). In keeping with this, we find that mucinspurified from some carcinomas also have binding sites for all three selectins.Direct competition studies indicate that the binding sites for each selectin alongthe mucin backbone are almost completely separate from one another. Thus, thespecificity of each selectin for binding carcinoma mucins seems to be dictatedby distinct combinations of O-glycans on the apomucin polypeptide backbone.These multiple distinct selectin binding sites along the length of the extendedmucin molecules could potentially mediate pathological interactions with andamongst leukocytes, platelets and endothelial cells. Indeed, we have recently
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demonstrated that the purified carcinoma mucins can mediate four suchpathological interactions: facilitating aggregation of partially activated plateletsin a P-selectin dependent manner; causing direct interaction of activatedplatelets with activated endothelium by bridging between P-selectin onmononuclear cells and E-selectin on activated endothelium. Further work isneeded to identify which of these and any other possible combinations ofinteractions actually occurs in vivo.
To obtain more direct in vivo evidence for the importance of selectins intumor biology, we studied the behavior of human colon adenocarcinoma cells(LS180, T84) in RAG2 -/- mice in the presence or absence of a P-selectindeficient background. We found a significant attenuation of both tumor growthand metastasis in the setting of P-selectin deficiency. Three potentialpathophysiological mechanisms have been demonstrated so far: first,intravenously injected tumor cells home to lungs of P-selectin deficient mice at alower rate; second, P-selectin-deficient mouse platelets fall to adhere to tumorcell surface mucins; finally, tumor cells lodged in lung vasculature afterintravenous injection are found to be decorated with platelet clumps, and theseclumps are markedly diminished in P-selectin deficient animals. The P-selectindependent tumors-platelet interaction seems to be very early evident, since it canbe detected within 10 min of intravenous injection of tumor cells. We arecurrently exploring if any other blood cell types are involved in thesecomplexes.
We also have preliminary results from syngeneic models for tumormetastasis in mice. These data indicate that the role of P-selectin in cancermetastasis is not only specific for human tumors, but also applies to murinetumors. Further studies of the potential role of other selectins in tumor growthand progression are currently under way.
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Ajit Varki, M.D.
Professor of MedicineUCSD Cancer CenterUniversity of California San DiegoLa Jolla, CA 92093-0687
1974 -75 Rotating Internship, Christian Medical College Hospital, Vellore1976 Research Asst. Biochem. Research Labs, Univ. of Nebraska
Medical Center, Omaha1976 - 77 Resident, Internal Medicine, Episcopal Hospital, Temple
University, Philadelphia1977 - 78 Resident in Internal Medicine, Univ. of Nebraska Medical
Center, Omaha1978 - 82 Fellow in Hematology/Oncology, Dept. of Internal Medicine,
Washington University School of Medicine, St. Louis(Laboratory of S. Kornfeld)
1980 - 82 Instructor in Medicine, Washington University School of Medicine,St. Louis
1982 - 87 Assistant Professor of Medicine, University of California, SanDiego
1987 - 91 Associate Professor of Medicine, University of California, SanDiego
1991 - pres. Professor of Medicine, University of California, San Diego1992 - 96 Editor-in-Chief, Journal of Clinical Investigation1993 - pres. Member, Division of Cellular and Molecular Medicine, UCSD1994 - 1997 Associate Director for Basic Research, UCSD Cancer Center1996 - 1997 Interim Director, UCSD Cancer Center
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Modification of Cell Surface Sialic Acid
Akiko KanamoriProgram of Experimental Pathology, Aichi Cancer Center Research Institute,Kanokoden, Chikusa-ku, Nagoya 464-8681 Japan
Most of sialic acids are bound at the nonreducing end of theoligosaccharides of the cell surface glycolipids or glycoprotemis. Modificationof sialic acids, such as N-acetyl hydroxylation and O-acetylation, increase thestructural diversity of sialoconjugates and contribute to their variety ofbiological functions.
The most common modification of the hydroxyl groups of sialic acids isthe addition of O-acetyl esters. There exist two types of O-acetylation. One is atC-4 of the pyranose ring and the other is at the glycerol side-chain of sialic acidat C-7,8,9. 4-O-acetylation causes completely resistant towards the action ofalmost all sialidases, but the modification is found in the limited species. Incontrast, there are more reports about the wide spread of the O-acetylation at theside-chain. In the case of 9-O-acetylation, the resistance towards the sialidasesseems to be weaker than that of 4-O-acetylation. But by the addition of anotheracetyl residues at C-7 and 8, the resistance becomes stronger. The retardation ofsialic acid degradation leads to a longer life time of glycoconjugate and cellswith side-chain O-acetylated sialic acid.
More evidence is accumulating the O-acetylation of sialic acid is apotent regulator of cellular adhesion. The three sialoadhesins, myelin-associatedglycoprotem, the macrophage sialoadhesin, and CD22 are all hindered to bind tosialic acid if they are 9-O-acetylated. In the other hand, the extent of O-acetylation decreases in the mucin from human colon tumor concomitantly withthe grade of malignancy and metastatic potential. This decrease of O-acetylationcauses the consistent overexpression of sialyl Lewis X antigen. The relationshipbetween the metastasis of carcinoma and the overexpression of selectin-ligand issuggested.
In spite of its importance, the O-acetylation mechanism is poorlyunderstood at the molecular and genetic levels because of the complexity andthe difficulty of the reconstitution of the mechanism. Recently, by expressioncloning method, we have isolated a cDNA, termed AT-1, encoding a novelprotein required for the formation of O-acetylated gangliosides. The cDNAencodes a protein with multitransmembrane spanning domains, termed AT-1-p.The amount of O-acetylated gangliosides increased significantly by
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overexpression of AT-1-p. When semi-intact cells prepared by treatment withstreptolysin O were incubated with [Ac-14MC]-acetyl coenzyme A (Ac-CoA),increased incorporation of radioactivity was found in those cells AT-1-poverexpressed when compared with the mock transfectants. The incorporationactivity was inhibited by the existence of CoA. These results suggest that AT-1-pis to be an Ac-CoA transporter that is involved in the process of O-acetylation.
It seems that the O-acetylation of sialic residues occurs in the Golgiapparatus. There should be a particular system to transport Ac-CoA synthesizedin the cytoplasm into the Golgi apparatus. Immunohistochemical study with anantibody specific to the AT-1-p suggested that it is most probably expressed inthe endoplasmic reticulum, membrane. Northern blot analysis showed two majortranscripts m all tissues examined to indicate that AT-1-p is a ubiquitousmolecule. Although AT-1-p seems to be an Ac-CoA transporter playing a keyrole in acetylation process other than that for sialic acid in gangliosides, furtherstudies will be required to prove its function.
Introduction of molecular biological technique brings us finding of newfactors included in the modification of sialic acid and speedup, of theinvestigation.
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Akiko Kanamori, Ph.D.
Program of Experimental Pathology,Aichi Cancer Center Research InstituteKanokoden, Chikusa-ku, Nagoya 464-8681, JapanPhone: 052-762-6111, ext. 8815Fax: 052-763-5233Email: [email protected]
1987 - 1990 Graduate student of Department of Biophysics and Biochemistry,Faculty of Science, The University of Tokyo (Ph.D.).
1990 - 1991 Fellowship of Hayashi Memorial Foundation for Female NatureScientists (research student of Department of Biophysics andBiochemistry, Faculty of Science, The University of Tokyo).
1991 - 1993 Fellowship of the Japan Society for the Promotion of Sciencefor Japanese Junior Scientists (at Dr. Yasuo Inoue's laboratoryin The University of Tokyo).
1993 - 1998 Research worker of Laboratory for Glyco-Cell Biology inFrontier Research Program, RIKEN
1994.4- 1994.10 Visiting Investigator of La Jolla Cancer Research Foundation(at Dr. Minoru Fukuda's laboratory)
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Dynamic O-Glycosylation of the Estrogen Receptor, TumorSuppressors and Oncogene Proteins: A Potential Role forO-GlcNAc in Oncogenesis.
Gerald W. Hart, Robert N. Cole, Lisa K. Kreppel, C. Shane Arnold, Frank I.Comer, Sal Iyer, Xiaogang Cheng, Jill Carroll, Glendon J. Parker, KeithVosseller, and Lance WellsDepartment of Biological Chemistry, Johns Hopkins University School ofMedicine, 725 N. Wolfe St., Baltimore, MD 21205-2185; Fax: (410) 614-8804;email: [email protected]
We have described a highly-dynamic form of protein glycosylation inwhich single N-acetylglucosamine monosaccharides are attached to Ser(Thr)hydroxyl moieties (termed O-GlcNAc; for review see Ann. Rev. Biochem. 1997.66:315-335). O-GlcNAc is similar to O-phosphorylation in most respects. Thesaccharide modification is apparently as abundant on nuclear and cytoskeletalproteins of virtually all eukaryotes as is phosphorylation. O-GlcNAc oftenoccurs at sites that are also used by kinases, and has a "yin-yang" relationshipwith phosphate on certain proteins. Proteins identified to date that bear O-GlcNAc include, many transcription factors and RNA polymerase II,translational regulatory proteins, kinases, tyrosine phosphatases, nuclear poreproteins, cytoskeletal regulatory and 'brifging proteins', viral and parasiteproteins. We hypothesize that O-GlcNAc is a regulatory modification whichmay play a role in transiently 'capping' phosphorylation sites and in regulatingprotein-protein interactions within the cell.
Of particular relevance to cancer are the findings that ligand-dependentnuclear transcription factors, such as the estrogen receptor, and many nuclearoncogene proteins or tumor suppressors are dynamically modified by O-GlcNAcylation, as well as by O-Phosphorylation. Site mapping studies of the c-Myc oncogene (J.Biol.Chem. (1995) 270, 18961-18965) indicate that O-GlcNAcand O-Phosphate compete for the same site (Thr58), which is also the majormutational "hotspot" in human Burkitt's lymphomas. In addition, hierarchicalphosphorylation of Thr58 and Ser62 have been shown to play an important rolein both the transcriptional and oncogenic properties of the protein. Recentstudies with synthetic glyco- and phospho-peptides indicate that the twomodifications influence the addition of each other in this key region.
The retinoblastoma proteins Rb and p107 also appear to be O-GlcNAcylated. Interestingly, our early studies indicate that O-GlcNAcylation of
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Rb is increased in the presence of the general kinase inhibitor, staurosporine.Current data supports the notion that glycosylated Rb interacts with tumorsuppressors and phosphorylated Rb forms do not. Similar studies have alsorecently shown that the p105 form of the key transcription factor regulatinglymphocyte growth and functions, NFkappaB is also O-GlcNAcylated. Wehypothesize that O-GlcNAc might play a role in regulating the conversion ofp105 to its active form, p50, which interacts with the rel family member p65 toform the functional dimer of the transcription factor.
O-GlcNAc sites have been mapped on the estrogen receptor (ER)purified from several sources (J.Biol.Chem. (1997) 272, 2421-2498). Sitemapping indicates that several sites are present in the F domain and at least onesite appears to be present in the DNA-binding domain of the receptor. Teflon-DNA binding affinity chromatography using the estrogen response element(ERE), indicates that the DNA-binding forms of ER are modified by O-GlcNAc.Site-directed conversion of the O-GlcNAc site at Thr575 in the F-domain,indicates that the saccharide might play a role in ER dimerization. ERE-gel shiftanalyses indicate that the DNA-binding forms are all "supershifted" by addingsWGA, a lectin specific for GlcNAc moieties. We have also shown that thenewly discovered estrogen receptor β is also extensively modified by O-GlcNAc.
The presence of dynamic O-GlcNAcylation on many key nuclearoncogene proteins and on key tumor suppressors or growth regulatorytranscription factors, suggests that drugs that specifically inhibit the addition orremoval or O-GlcNAc might provide new avenues of cancer therapy. Recently,we have cloned one of the O-GlcNAc transferases, which is a highly conservedtetratricopeptide repeat containing protein (J. Biol. Chem. (1997) 272, 9308-9315). We have also purified (J. Biol. Chem. (1994) 269, 19321-19330) andtentatively cloned an open-reading frame that appears to be an O-GlcNAcase.Having these clones will greatly facilitate elucidation of the regulation of O-GlcNAcylation. (Supported by NIH CA42486, HD13563, a grant from theJuvenile Diabetes Fdn., and by a grant from the Alzhemer's Disease ResearchProgram of the American Health Assistance Foundation.)
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Gerald W. Hart, Ph.D.
DeLamar Professor & Director of Biological ChemistryJohns Hopkins University, School of Medicine
Born 7/16/1949, Topeka, KS1971 - B.S. Biology and Chemistry, Washburn Univ., Topeka, KS1977 - Ph.D. Developmental Biology, Kansas State University,
Manhattan Kansas1977 - 1979: Jane Coffin Childs Postdoctoral, Dept. Biol. Chem. Johns
Hopkins Univ. Sch of Medicine1979 - 1984: Assistant Professor of Biological Chemistry, Johns Hopkins
University School of Medicine1984 - 1988: Associate Professor of Biological Chemistry, Johns Hopkins
University School of Medicine1988 - 1993: Professor of Biological Chemistry, Johns Hopkins University
School of Medicine1993 - 1997: Chair, Department of Biochemistry and Molecular Genetics,
Univ. of Alabama Sch. of Med.1997 - Present: Director & DeLamar Professor of Biological Chem., Johns
Hopkins University Sch. of Med.Memberships: ASBMB, ASCB, Soc. for Glycobiology, Amer. Chem. SocietyMajor Honors & Awards: First Intl. Glycoconjugate Organization AwardRecipient (1997); 1995-1998- Council of the ASBMB. 1995 - President, Societyfor Glycobiology; 1991- Dean's Lecturer: Johns Hopkins University MedicalSchool; 1983-1988 - Established Investigator, American Heart Association; 1975Recipient of the H.H. Haymaker Award for Excellence in Graduate StudentResearch.
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MEMO
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Circulating Autoantibody to Estrogen Receptor inEstrogen-dependent Cancer
Masaru Ishii and Kensei YamaguchiDivision of Gastroenterology, Saitama Cancer Center, Saitama, Japan
Overexpression of estrogen receptor (ER) and mutations in the ERmRNA in estrogen-dependent human cancer such as human breast cancer werewell known already. On the other hand, the first evidence of circulatingautoantibodies to ER in normal human serum was reported in 1987 by Mudarris,A. and Peck, E.J. From their report, it is speculated that increasing ofextracellular ER-antigen resulted in destruction of cancer cells caused by host-immune reaction enhances production of the autoantibodies to ER. However, theoccurrence of autoantibodies to ER in serum from patients with estrogen-dependent cancer is not yet reported.
In the present study, circulating autoantibodies to ER in sera fromestrogen-dependent cancer patients such as patients with breast cancer, prostatecancer and hepatocellular carcinoma (HCC) were examined and usefulness as atumor marker of these estrogen-dependent cancer was evaluated. Furthermore,immunohistological study using three kind of anti-ER rabbit antibody to eachdifferent antigenic determinant was performed compared cancerous tissues withadjacent non-cancerous tissues in mammary glands, prostate glands and liverand ER antigenic determinant which is specific to estrogen-dependent cancerwas investigated.
A solid phase enzyme-immunoassay (EIA) sandwiching anti-ERbetween ER-peptide antigen bound to surface of microplate wells and biotin-labeled ER-peptide antigen was developed for the determination of serum anti-ER. The ER-peptide antigen employed for the assay was a 20 mers syntheticpeptide that correspond to an ER-antigenic determinant in the domain D of ER.Rabbit IgG antibody to ER-peptide antigen was used as a standard ER antibodyin the assay. By the EIA, antibodies to ER in serum samples from normal donors,patients with benign breast disease, patients with benign prostate hypertrophyand patients with chronic hepatitis, breast cancer patients, prostate cancerpatients and patients with HCC was measured.
Serum anti-ER value in 20 normal donors showed level under 25 ng/mland no significant difference between 12 males and 8 females. One (5%) of 20normal donors, 1 (25%) of 8 patients with benign breast disease, 11 (77%) of 13
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patients with prostate hypertrophy and all (100%) of 3 patients with chronichepatitis showed serum anti-ER value beyond 20 ng/ml. On the other hand, theanti-ER value over 20 ng/ml was seen in 23 (46%) of 50 patients with breastcancer, 2 (14%) of 14 patients with prostate cancer and 8 (73%) of 11 patientswith HCC. From above results, circulating autoantibodies to ER suggested to beavailable as a tumor marker of breast cancer for female.
Immunohistological examination was performed using 3 kind of anti-ER rabbit antibody to each ER-antigenic determinant in the domain A/B, D andF of ER. These anti-ER antibodies stained both of cancerous tissues and adjacentnormal tissues in the mammary glands, respectively. The cancerous tissues oradjacent normal tissues in the prostate glands reacted against anti-ER to thedomain A/B or F, but not anti-ER to the domain D. In the fiver tissues both ofcancerous tissues and adjacent cirrhotic tissues was stained with these anti-ERantibodies, respectively. Localization of ER judged from anti-ER-stained cellswas only nuclear form in the mammary glands and prostate glands, but eithernuclear or cytoplasmic form in HCC or cirrhotic liver tissues. Furthermore, asurvey of ER mRNA by RT-PCR method for HCC and cirrhotic liver tissuesresulted in obvious demonstration of the expression of ER mRNA.
Above immunohistological results that the expression of each domainin the ER might be influenced by kind of organ and cellular status of cancer ornon-cancer may contribute to development of a novel tumor marker forestrogen-dependent cancer, following elucidation of the ER-antigenicdeterminant which is organ- and cancer-specific.
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Masaru Ishii, M.D.
Division of GastroenterologySaitama Cancer Center818 Komuro, Ina-Machi, Kitaadachi-Gun,Saitama, 362-0806, Japan
1962 Kobe University School of Medicine, Kobe (MD)1962-1963 Internship, Kobe University School of Medicine1963-1974 Department of Second Internal Medicine, Kobe University
School of Medicine1974-1981 Head, Division of Gastroenterology Saitama Cancer Center1981-1991 Director, Department of Laboratory Medicine, Saitama Cancer
Center1991-1993 Deputy Director of Hospital, Saitama Cancer Center1993-1993 Director of Research Institute, Saitama Cancer Center1993-1998 Director of Hospital, Saitama Cancer Center1998-present President, Saitama Cancer Center
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Molecular Basis of Leukocyte Recognition of EndothelialSelectins
Geoffrey S. Kansas, PhDDept of Micro-Immuno, Northwestern University Medical School, 303 E.Chicago Ave., Chicago, IL 60611, USA.
The selectins are a group of carbohydrate-binding adhesion moleculesexpressed on leukocytes (L-selectin), activated endothelium (E- and P-selectin),or activated platelets (P-selectin). As such, selectins mediate adhesiveinteractions between leukocytes and other leukocytes, endothelium, andactivated platelets. These interactions are therefore crucial to lymphocytehoming, leukocyte recruitment in inflammation, hemostasis, and metastasis ofblood-borne tumor cells. We have been interested in defining the enzymatic andstructural basis for recognition of E- and P-selectin by human leukocytes.
Several lines of evidence definitively identify a homodimericglycoprotein designated PSGL-1 as the principal or sole ligand for P-selectin onleukocytes. First, monoclonal antibodies (mAb) against PSGL-1 block adhesionof all normal leukocytes as well as cell lines and transfectants to P-selectin, butdo not affect binding to E-selectin. Second, PSGL-1 negative cells and cell linesdo not bind to P-selectin. Third, binding to P-selectin is conferred bytransfection of cell lines of appropriate glycosylation phenotype with PSGL-1cDNA, and the binding of these transfectants is blocked by PSGL-1 mAb.Fourth, proteases which cleave PSGL-1 block binding to P-selectin, but do notaffect binding to E-selectin. Collectively, these findings argue strongly for anessential role for PSGL-1 in leukocyte binding to P-selectin.
PSGL-1 is also a major ligand for L-selectin. As such, PSGL-1 plays animportant role in mediating leukocyte-leukocyte interactions, includingneutrophil aggregation, which may play an important role in amplifyingleukocyte recruitment at sites of inflammation. In several in vitro systems, amajor portion of leukocyte recruitment was dependent on these so-called"secondary" interactions. In vivo, these secondary interactions are likely to playan important role in smaller vessels, including those that typically support themajority of leukocyte recruitment.
We and others have identified several structural features of PSGL-1which play essential roles in adhesion to PSGL-1. Mutational analysis hasdemonstrated that sulfation of at least one of a cluster of three tyrosines
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contained within a consensus tyrosine sulfation motif at the extreme aminoterminus of the mature, PACE-cleaved protein is essential for recognition ofboth P-selectin and L-selectin. A mAb (designated KPL1) whose epitope mapsprecisely to this region, and which requires sulfation for biding, abolishesrecognition of P- and L-selectin. The existence of a sulfate moiety as part of thenatural ligand for P-and L-selectin on leukocytes is one of several featurescommon to ligands for L-and P-selectin.
Mutational analysis has also identified Thr15, only a few residues downfrom the tyrosine sulfate motif, as essential for binding of PSGL-1 to P-selectin.It is likely that the carbohydrate structures synthesized by the enzymes FucT-VIIand C2GnT, discussed below, are attached to this residue. The precisecarbohydrate structure(s) required for recognition of P- (and L-) selectin havenot yet been precisely defined. In addition, how carbohydrates attached to Thr15interact with sulfate moieties just upstream to form the physiologic binding siteis not yet clear.
We have recently focused on the dimerization of PSGL-1. We showedthat dimerization is mediated by Cys320, one of three cysteines in the molecule,all of which are conserved in the mouse. Cys320 is predicted to be located justoutside the membrane, whereas the others are either within the transmembraneregion or in the cytoplastmic tall. Mutation of cys320 to alanine (C320A) blocksdimerization of PSGL-1 at the cell surface, as assessed by cross-linking studiesand native gel electropheresis, and also blocks adhesion to P-selectin. The basisfor this requirement for dimerization is presently under investigation, but mayinvolve creation of a binding site in trans, between carbohydrate moietiesattached to Thr15 and sulfates within the tyrosine sulfation motif.
The enzymes which carry out the post-translational modifications ofPSGL-1 and other selectin ligands have been the focus of study of severalgroups, including our own. A tyrosine sulfotransferase which is responsible forsulfation of PSGL-1 has recently been identified and cloned. The O-linkedbranching enzyme, core-2 β1,6 N-acetyglucosaminyltransferase (C2GnT) hasbeen shown to be required for appropriate modification of PSGL-1. In addition,the leukocyte α1,3 fucosyltransferase FucT-VII has been shown to be requiredfor construction of ligands for all three selectins, including PSGL-1. In T cells,where expression of FucT-VII is inducible and regulated, very low levels ofFucT-VII are sufficient for maximal binding to P-selectin, whereas higher levelsare required for binding to E-selectin. We have also found that T cells activatedin the presence of IL-12, a Th1-polarizing cytokine, have greatly enhancedlevels of FucT-VII compared to T cells activated under non-polarizing
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conditions, whereas T cells activated in the presence of IL-4 have significantlylower levels of FucT-VII. In contrast, levels of C2GnT are increased by T cellactivation regardless of the cytokines present. This selective control of FucT-VIIby T cell polarizing cytokines underlies the selective ability of Th1 cells to hometo sites of inflammation in vivo.
With regard to E-selectin ligands, the situation is more complex.Although various molecules have been proposed as E-selectin ligands, includingL-selectin and PSGL-1, these molecules are neither necessary nor sufficient forbinding to E-selectin. MAb against L-selectin or PSGL-1 do not block directbinding to E-selectin, and transfection of cells with cDNA encoding thesemolecules does not confer or enhance binding to E-selectin. However, enforcedexpression of FucT-VII is sufficient to confer strong binding to E-selectin of anycell type tested, including non-hematopoietic cells, regardless on theglycosylation phenotype of the cell. In particular, C2GnT is not required forstrong binding to E-selectin. Also unlike P-selectin, expression of FucT-IV, asecond leukocyte αl,3 fucosyltransferase, is sufficient in some cell lines forbinding to E-selectin. Binding to E-selectin does not require sulfation. Takentogether, these differences in glycosyltransferase requirements argue that ligandsfor the two endothelial selectins are quite distinct.
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Geoffrey S. Kansas
HOME ADDRESS: 285 Wilmot Road, Deerfield, IL 60015, (847) 374-0145WORK ADDRESS: Department of Microbiology-Immunology, North-western
Medical School, 303 East Chicago Avenue, Chicago, IL60611, (312) 908-3237, email <[email protected]>
DATE OF BIRTH: January 22, 1957PLACE OF BIRTH: Philadelphia, PAEDUCATION:1978 B.A. Brandeis University, Waltham, MA (Biology)1984 Ph.D. Stanford University, Stanford, CA (Cancer Biology)POSTDOCTORAL TRAINING:2/1994-7/1994 Research Associate, Division of Hematologic Malignancies,
Dana Farber Cancer Institute (Laboratory of James D.Griffin, M.D.) and Department of Pathology, HarvardMedical School, Boston, MA
2/1990-7/1993 Research Associate, Division of Tumor Immunology, DanaFarber Cancer Institute (Laboratory of Thomas F. Tedder,Ph.D.), and Department of Pathology, Harvard MedicalSchool, Boston, MA
7/1986-1/1990 Postdoctoral Fellow, Department of Pathology (Laboratoryof Morris O. Dailey, M.D., Ph.D.), University of IowaCollege of Medicine, Iowa City, IA
8/1985-6/1986 Research Scientist, ORTHO Pharmaceutical Corp., Raritan,NJ
9/1984-7/1985 Postdoctoral Fellow, Stanford University School ofMedicine, Department of Pathology (Laboratory of EdgarG. Engleman, M.D.), Stanford, CA
HONORS:NIH predoctoral fellowship T32-CA09119, 1979-1984; NIH postdoctoralfellowship 1F32-AI07820, 1987-1989; Associate Editor, Journal of Immunology,1995-; ad hoe reviewer for: Journal of Immunology, 1988-; Blood, 1990-;Journal of Cell Biology, 1994-- Journal of Clinical Investigation, 1997-; Journalof Biological Chemistry, 1997-; Journal of Experimental Medicine, 1997-; ProcNatl Acad Sci, 1997-,FEDERAL GOVERNMENT PUBLIC ADVISORY COMMITTEE SERVICE:IVP Study Section, 6/1996; 10/1996 (ad hoc); Pathobiochemistry Study Section,2/97 (outside).
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L-Selectin Ligand: Molecular Cloning and Characterizationof an N-Acetylglucosamine 6-O-Sulfotransferase Involved inSynthesis of 6-Sulfo Sialyl Lewis X
Takashi Muramatsu1, Kenji Uchimura1, Naoko Kimura2, Hideki Muramatsu1,Qi- Wen Fan1, Chikako Mitsuoka2, Akiko Kanamori2, Nozomu Hiraiwa2, KenjiKadomatsu1, Nobuyuki Kurosawa1, Taishi Yamakawa1, Osami Habuchi3 andReiji Kannagi2
Department of Biochemistry, Nagoya University School of Medicine1, Japan,Program of Experimental Pathology2, Aichi Cancer Center, Research Institute,Japan, Department of Life Science3, Aichi University of Education, Japan
Sulfated sialyl Lewis X structures have been implicated as the ligand ofL-selectin. Immunochemical, chemical and enzymological studies strongly favorthat 6-sulfo sialyl Lewis X is the ligand. Based on sequence homology tochondroitin-6-sulfotransferase, we cloned mouse and human N-acetylglucosamine-6-O-sulfotransferase. The mouse and human enzymes werehighly homologous in the 363 amino acid stretch of the putative catalytic region;the two proteins were nearly identical except for 3 conservative changes.
The enzyme expressed in COS 7 cells transferred sulfate to the 6position of non-reducing GlcNAc in GlcNAc β 1-3Gal β 1-4GlcNAc andGlcNAc β 1-3Gal β 1-4GlcNAc β 1-3Gal β 1-4GlcNAc. Co-transfection of theenzyme cDNA and fucosyltransferase VII (FucT VII) cDNA into COS-7 cellsresulted in cell-surface expression of 6-sulfo sialyl Lewis X. In situhybridization verified the presence of the enzyme mRNA in the high endothelialvenules of mesenteric lymph nodes. Therefore, the enzyme is likely to beinvolved in synthesis of the L-selectin ligand. Indeed, functional L-selectinligands could be reconstituted on cultured human endothelial cell line ECV 304by double transection with the sulfotransferase cDNA and FucT VII cDNA.Significant adhesion of L-selectin expressing cells were seen only to the doublytransected cells.
mRNA of the human enzyme was strongly expressed in the bonemarrow, peripheral blood leukocytes, spleen, brain, spinal cord, ovary, andplacenta, and moderate levels of expression were observed in many organsincluding lymph nodes and thymus. Among human tumor cells, strongexpression of the mRNA was found in MOLT-4 and Jurkat lymphoblasticleukemia cells, Raji lymphoma cells, K-562 chronic myelogenous leukemiacells, U 251 glioma cells and G361 melanoma cells. In addition to the role as the
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L-selectin ligand, carbohydrate structures synthesized by the sulfotransferasemay be involved in various aspects of the differentiation and behavior of bloodcells and their progenitor cells.
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Takashi Muramatsu, Ph D
Department of BiochemistryNagoya University School of Medicine65 Tsurumai-cho, Showa-kuNagoya 466-8550 Japan
1963 Department of Biophysics and Biochemistry, Faculty of Science,Tokyo University (BS)
1968 Department of Biophysics and Biochemistry, Faculty of Science,Tokyo University (PhD)
1968-1972 Research Fellow, Department of Microbiology andImmunology, Albert Einestein College of Medicine
1972-1973 Assistant Professor, Department of Biochemistry, KobeUniversity School of Medicine
1973-1980 Associate Professor, Department of Biochemistry, KobeUniversity School of Medicine
1977-1978 Visiting Scientist, Department of Molecular Biology, PasteurInstitute
1980-1993 Professor, Department of Biochemistry, Faculty of Medicine,Kagoshima University
1993-Present Professor, Department of Biochemistry, Nagoya UniversitySchool of Medicine
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Transcriptional Regulation of Selectin Ligand Expressionin ATL Cells
Nozomu HiraiwaLaboratory of Experimental Pathology, Aichi Cancer Center Research Institute,Nagoya, Japan
The human Fuc-T VII (hFuc-T VII) gene encodes aleukocyte/endothelial cell adhesion molecule (Selectin)-ligand sialyl LeX
synthase, the expression of which is regulated during the hematopoieticdifferentiation, inflammation and malignant transformation. Recent studiesrevealed that the synthesis and expression of sialyl Lewis X and selectin ligandsin leukocytes and in leukemic cells are controlled principally at the level oftranscription of the FucT VII gene. We have previously shown that leukemiacells in patients with adult T-cell leukemia (ATL), and cell lines derivedtherefrom, strongly express the sialyl Lewis X, relevant to transcriptional levelof the Fuc-T VII gene, compared to other types of human lymphocytic leukemiacells. ATL is an aggressive and fatal malignancy of T-helper cells. ATL ischaracterized by the vigorous extravascular infiltrative activity of the leukemiacells, which adds distinct clinical features to this disease, such as the infiltrationof the skin by leukemia cells. The degree of expression of sialyl Lewis X onleukemia cells in ATL significantly correlates with the degree of extravascularinfiltration of the leukemia cells. These evidences suggest that the sialyl LewisX determinant expressed on leukemia cells is involved in the infiltration ofleukemia cells from blood vessels to tissues and organs, since the binding ofsialyl Lewis X on leukocytes and E-, or P-selectin on endothelial cells is knownto trigger the extravasation of leukocytes. In addition, expression of sialyl LewisX and transcripts of hFuc-T VII gene is induced by human T cell lymphotrophicvirus type I Tax protein, which has a profound transactivating effect on a widevariety of important cellular genes. So it is important to investigate howtranscription of hFuc-T VII gene is regulated and how Tax protein induces thetranscription in leukemic cells.
In order to understand how expression of Fuc-T VII gene is controlled,we first determined the genomic organization of hFuc-T VII gene andcharacterized its promoter. We have isolated genomic clones of the gene,analyzed the genomic organization, and begun to characterize Fuc-T VII gene
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promoter. Then next, we made a survey of the transcription level amongleukocyte and leukemic cell lines and found that most cell lines derived from B-cells and in immature T-cell lines including Jurkat T cells hFuc-T VII is littletranscribed. On the contrary the YT cells, NK-like cell line, the HL-60 cells,promyelocytic leukemia cell line, and the ED40515-N cells, ATL cell lineexpressing Tax, all of them express Fuc-T VII gene constitutively. 5'-RACE andprimer extension analyses identified multiple transcriptional start pointscentering on a motif homologous to the initiation element (Inr), and the majortranscription start point located at 139 nucleotides upstream relative to thetranslational initiation site. Reporter assays revealed basal promoter activity inYT cell line, which highly expresses the hFuc-T VII gene constitutively. We alsoanalyzed promoter activity in Jurkat T cells, which hardly transcribe the hFuc-TVII gene, but deliver its induction upon TPA-stimulation or with co-expressionof Tax protein. Deletion analyses demonstrated that the minimal promoteractivity could be modulated by various sequences within 1-kilobase 5'-flankingregion. The minimal promoter was embedded in a GC-rich domain (73%, GCcontent), in which one Sp1 binding motif as well as an Inr were found, but itlacked TATA and CAAT boxes. Inactivation of the Sp1 site abolished basaltranscription in those cell lines and phorbol ester-induced transcription in JurkatT cells. The positive regulatory region located between -193 and -134 from themajor transcription start point, contained activating transcription factor (ATF)-like sequence, where consensus CRE sequence inhibited DNA-protein complexlike transcription factors of ATF family. Mutation of the site significantlyreduced the promoter activity stimulated by phorbol ester, which suggests thissequence acts on transcription as TRE (TPA-responsive element). The TATA-less promoter region of the hFuc-T VII probably thus controls the cell type-specific and phorbol ester-induced expression of the hFuc-T VII genesignificantly via this site (called "TRE/ATF-like sequence"). Surprisingly, co-transfection of Tax-expressing plasmids showed that this "TRE/ATF-like" sitecontributes significantly to induction of the hFuc-T VII transcripts by Tax. Thispotential role of the CREB/ATF transcription factors in Tax-mediatedtransactivatin via this "TRE/ATF" site was confirmed by analyzing theinvolvement of Tax mutants that functionally segregate several pathways of Tax-mediated trans activation. Reporter assay in Jurkat T cells expressing a Taxmutant selectively deficient in the ability to activate transcription throughCREB/ATF demonstrated marked decrease in promoter activity of the hFuc-TVII gene, compared to other kinds of Tax mutants. To make sure of associationof Tax with this site, the DNA-protein complexes were analyzed by
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immunoblotting with anti-Tax through DNA-affinity precipitation (DNAP)assay. When the wild-type "TRE/ATF-like" sequence was use, Tax protein wasdetected in the sequence-protein complexes in the nuclear extract, but was notdetected with use of a mutant of the sequence. This observation suggests thesignificance of the complexes mi the transactivation of the "TRE/ATF-like"enhancer site by Tax protein, and this "TRE" also means Tax responsive elementas well as TPA-responsive element.
This situation really resembles gene expression from HTLV-1 long terminalrepeat (LTR) mediated by the 21-bp repeats and Tax. In addition to functionalsimilarities, the sequence of the "TRE/ATF-like" site can be subdivided intothree motifs, quite similar to the HTLV-1 21-bp repeats. Through CRE-likemotif in this 21-bp repeats Tax potentiates transactivation of LTR in a differentmanner from activation of cellular CRE-containing genes by Tax. Activation ofcellular CRE in the presence of Tax is phosphorylation-dependent, but activationof the 21-bp repeats is phosphorylation-independent. In fact, Tax activatesexpression of the hFuc-T VII gene even in the absence of PKA, which suggeststhat Tax activates the hFuc-T VII "TRE/ATF-like" site through similarmechanism to viral CRE in the 21-bp repeats. Since Tax activation of the hFuc-T VII in that manner is the first evidence reported among endogenous genestransactivated through cellular CREs, it is very intriguing to investigate how thehFuc-T VII gene encoding a leukocyte/endothelial cell adhesion molecule(Selectin)-ligand sialyl LeX synthase, interacts cellular transcription factors inATL leukemic cells, and we expect that the finding obtained from this study willbe useful to treatment of ATL disease.
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Nozomu Hiraiwa, M.D.
Laboratory of Experimental PathologyAichi Cancer Center Research InstituteNagoya, Japan
1976-1982 Kyoto University School of Medicine, Kyoto (M.D.)1982-1985 Residency, Department of Internal Medicine, Toranomon
Hospital, Tokyo1985-1989 Kyoto University Graduate School of Medicine (D.M.Sci)1989-1990 Medical Staff, NTT Kyoto Hospital, Kyoto1990-1994 Research Associate, Howard Hughes Medical Institute,
University of Michigan, Ann Arbor MI1994-present Senior Research Staff, Laboratory of Experimental
Pathology, Aichi Cancer Center Research Institute
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Clinical Value of Genetic Detection for Rare Cancer Cellsin Circulation and Lymph Nodes
Shoji Nakamori, Yoshiyuki Fujiwara, Masao Kameyama*, Hiroshi Furukawa*,Yusuke Nakamura#, and Morito MondenDepartment of Surgery II, Osaka University Medical School, Osaka,*Department of Surgical Oncology, Osaka Medical Center for Cancer andCardiovascular Diseases, Osaka, and #Laboratory of Molecular Medicine,Institute of Medical Science, University of Tokyo, Tokyo, Japan
To date, most of the tumor stage classification systems are based on themorphological findings which are obtained by clinical information, tactile andvisual examination, or pathologic evaluations. They, however, are not alwaysaccurate in predicting patient's outcome because of the limited ability indetecting micrometastases. Molecular diagnosis are focused on to establishreliable and sensitive detection of the extent of tumor's spread and to estimateprognosis and in planning optimal management for patients with cancer.
The advent of polymerase chain reaction (PCR) has allowed thedetection of rare cancer cells in blood, bone morrow, and lymph node. We usedthe expression of the epithelial cell specific cytokeratin (CK) 19 mRNA as anindicator of cancer cells and examined its expression both in the peripheral or inthe tumor drainage vein blood obtained from 49 gastric cancer, 59 colorectalcancer, and 38 pancreatic cancer patients and in lymph nodes of 12 gastriccancer patients by the reverse transcriptase polymerase chain reaction (RT-PCR).Furthermore, to evaluate the prognostic significance of detection of occultcancer cells in regional lymph nodes by means of genetic alterations which arefrequently found in colorectal cancer, we retrospectively screened 50 patientswith stage II colorectal cancers for mutations in K-ras (codons 12,13, and 65) orp53 (exon 5-8) by Mutant-allele-specific amplification (MASA). MASA is atechnique that can detect, at the level of an individual cells, micrometastases inlymph nodes that are histologically diagnosed as negative.
CK19 RT-PCR products were detected at a concentration as low as oneto 10 cancer cells per 106 normal peripheral blood mononuclear cells in theserial dilution studies. However, CK19 RT-PCR products were detected in six of69 samples from healthy controls of the first assay and in five of the secondassay, while no sample was detected for CK19 RT-PCR product at the eitherassay. To avoid false positive result, sample was defined as positive for CKwhen the separate assays gave the accordant results. According to this criteria,
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positive rate for CK19 RT-PCR product in peripheral vein blood was none (0 %)of 49 gastric cancer, one (1.7%) of 59 colorectal cancer, and two (5.3%) of 38pancreatic cancer patients. In drainage vein blood, none (0%) of gastric cancer,nine (15%) of colorectal cancer, and two (5.3%) of pancreatic cancer patientswere also positive for CK19. None of the samples were immunocytologicallydetectable for CK19. Of 11 patients positive for CK19 in blood, nine patientshad tumors with distant metastases at the time of surgery.
CK19 RT-PCR product in lymph node was examined in 100 lymphnodes obtained from 12 gastric cancer patients. Seven lymph nodes which werepathologically metastasis-positive were also positive for CK19 RT-PCR product.Of the 93 lymph nodes which were pathologically metastasis-negative, 14 (15%)were positive for CK19, indicating that these lymph nodes containedmicrometastases which could not be detected by pathological examination. Fivepatients with CK19 positive lymph nodes had T2 or T3 tumors, while five of theseven remained patients with CK19 negative lymph nodes had Tis or T1 tumors.Of 27 stage II colorectal cancers in which somatic mutations were identified byMASA, 11 (41%) had the corresponding mutations in regional lymph nodespreserved after pathologic examination. There are no significant correlationbetween the presence of genetically positive lymph nodes and clinicopathologicfeatures such as age, sex, tumor location, histologic type, depth of tumorinvasion, and presence of vessel invasion. None of the 16 patients who wereMASA negative lymph nodes had a recurrence within 5 years after surgery,while 11 patients with MASA positive lymph nodes had recurred.
These findings suggested that cancer cells in circulation or lymph nodeswhich were not pathologically detected could be genetically detectable.However, at present, genetic diagnosis of micrometastases may not be alwaysapplicable for the clinical use. Further prospective study in a large scale ofpatients and with more specific techniques is required to establish the geneticdiagnosis as routine clinical procedure.
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Shoji Nakamori, M.D.
Assistant Professor,Department of Surgery II,Osaka University Medical School2-2 Yamadaoka, Suita,Osaka, 565-0871 Japan
1976- 1982 Osaka University Medical School (M.D.)1982- 1983 Resident, Department of Surgery II, Osaka University,
Medical School1983- 1985 Resident, Department of Surgical Oncology, Osaka Medical
Center for Cancer and Cardiovascular Diseases1985- 1989 Clinical Fellow, Department of Surgery II, Osaka University
Medical School and Research Fellow, Oncogene Research,Research Institute for Microbial Disease, Osaka Unliversity
1989- 1990 Chief Surgeon, Department of General Surgery,Nachikatsuura Municipal Hospital
1990- 1991 Research Associate, Department of Tumor Biology, TheUniversity of Texas, M.D. Anderson Cancer Center
1991- 1997 Chief Surgeon, Department of Surgical Oncology, OsakaMedical Center for Cancer and Cardiovascular Diseases
1997- present Assistant Professor, Department of Surgery II, OsakaUniversity Medical School
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Molecular Diagnostic Detection of Free Cancer Cells in thePeritoneal Cavity of Patients with Gastrointestinal andGynecological Malignancies
Hayao NakanishiLaboratory of Pathology, Aichi Cancer Center Research Institute, Nagoya,Japan
While not all free tumor cells present in body fluids survive, one couldpostulate that patients with free tumor cells might be candidates for adjuvantchemotherapy because of the high risk of relapse. In this study, even moresensitive detection of free tumor cells could be achieved through amplificationof tissue specific mRNA by means of the reverse transcriptase-polymerase chainreaction (RT-PCR).
Peritoneal dissemination is the most frequent type of recurrence aftercurative resection in patients with gastric and ovarian cancers in Japan andWestern countries. Free cancer cells derived from serosal or capsular invasionmight be an indicator of early peritoneal seeding with subsequent formation ofmetastatic colonies. Their detection, therefore, is likely to be a useful tool forprediction of outcome in such cases. Cytological examination of peritonealwashes has been a Gold standard for assessment of peritoneal recurrence ingastric and ovarian cancer patients. Nevertheless, some of the patients withnegative cytology results die of peritoneal metastases after curative surgery. Inthis paper, very sensitive molecular diagnostic detection of free cancer cells inthe peritoneal cavity is reported and its prognostic relevance discussed.
RT-PCR analysis with primers specific for carcinoembryonic antigen(CEA) and conventional cytology examination were performed on peritonealwashes, collected at laparotomy from 199 gastric carcinoma patients. RT-PCRwas found to be more sensitive than cytology examination for detection of freetumor cells in the peritoneal washes, with higher detection rates for each of theT-categories in the TNM classification. Six patients with synchronous and 3 withrecurrent peritoneal dissemination were found among 18 advanced cancerpatients with positive PCR and negative cytology results. None of the patientswith negative PCR demonstrated intraperitoneal recurrence. Positive PCRresults were significant findings associated with poor survival of patients withadvanced gastric carcinomas (p<0.002).
RT-PCR analysis with primers specific for MUCI epithelial mucin were
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also performed on peritoneal washes from patients with ovarian cancer.Peritoneal washes of 16 of 21 ovarian carcinoma cases, including all of 13 withpositive cytology results, proved positive for MUCI mRNA, again indicating ahigher sensitivity with this method than conventional cytology.
One difficulty with molecular diagnostic techniques is that they aretime-consuming and relatively laborious as compared with conventionalmorphological methods. Recent advances in PCR technology, however, havemade it possible to dramatically reduce the time for amplification and detectionof mRNA. We have established a rapid and simple detection method for specificmRNA using the LightCyclerTM in combination with one step RT-PCR.Consequently, we could complete RT-PCR detection of CEA mRNA inperitoneal washing within 2 hours with essentially the same sensitivity.
Rapid and convenient detection of free cancer cells in peritoneal washes,most reliably by RT-PCR, is a powerful technique to predict peritonealdissemination m patients with gastric or ovarian cancers.
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Hayao Nakanishi, M. D.
Laboratory of Pathology,Aichi Cancer Center Research Institute,Kanokoden, Chikusa-ku,Nagoya 464,Japan
1979, MD. Ehime Univ. School of Med.1983, PhD. Ehime Univ. School of Med.1984-87, Researcher, Clinical Res. Inst. National Nagoya Hospital1988-91, Senior Researcher, Aichi Cancer Center Res. Inst.1992-93, Guest Researcher, National Institute on Aging, Baltimore1994- Section Head, Lab. of Pathology, Aichi Cancer Center Res.
Inst.
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Tissue-specific mRNA in Cancer Detected in PeripheralBlood by Reverse Transcriptase-Polymerase ChainReaction as a Novel Tumor Marker
Kazuhiko Uchida, M.D.Department of Biochemistry and Molecular Oncology, Institute of Basic MedicalSciences and Center for Tsukuba Advanced Research Alliance, University ofTsukuba, Tsukuba, Japan.
Sensitive diagnostic method in early stage of cancer, which is difficultto be found by imaging diagnostic devices such as computed tomography andultrasonography, has a major impact on the therapeutic strategies against cancerand an improvement of curability. Detection of mRNA for the cancer-relatedproduct in peripheral blood by reverse transcriptase-polymerase chain reaction(RT-PCR) is now a highly sensitive method to detect micrometastasis andcirculating cancer cells in the late clinical stages. Limitation of RT-PCR fortumor marker mRNA in cancer diagnosis is the false-positive result in normalcirculation because of a small amount of the tumor marker mRNA in blood cellsand high sensitivity of RT-PCR. Our purpose is to diagnose cancer and toidentify the suffered organ in early stage of cancer using circulating blood fromcancer patients. When we find cancer of unknown origin on screening by RT-PCR using circulating blood, tissue-specificity to identify the origin and tumor-specificity for discrimination from benign diseases are crucial. If a small amountof cancer cells circulate in patient's blood even in early stage and non-cancercells could not enter into vessel and circulate, not only detection of tumor-specific mRNA but also detection of tissue-specific mRNA in circulation by RT-PCR can indicate presence of cancer. Here, we screened "real" tissue-specificmRNA and established cancer detection method by RT-PCR using peripheralblood.
α-fetoprotein (AFP) and pro state- specific antigen (PSA) in serum arewidely used as tumor markers in evaluation of prognosis and management ofpatients with hepatocellular carcinoma and prostate cancer, respectively. Tissue-specificity of AFP and PSA were analyzed by Northern blot using poly A+ RNAsfrom 50 human tissues and RT-PCR using several cancerous tissues as well asnormal tissues and peripheral blood cells from healthy volunteers. As the results,broad expression of AFP was observed in several tissues as well as in fetal liver,and PSA was expressed mi salivary gland, pancreas and uterus other than
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prostate. By RT-PCR, AFP and PSA mRNA were detected in several tumorsincluding salivary pleomorphic adenoma, hilar bile duct carcinoma, pancreaticcarcinoma, transitional cell carcinoma of urinary bladder and thyroid papillarycarcinoma. Furthermore, both AFP and PSA mRNAs were frequently detectedby RT-PCR even in normal circulating blood. So that, we looked for issue-specific mRNAs in the functional organ like hormone-producing tissues, andfocused on the gene involved in production of thyroid hormone. Northernblotting using polyA+ RNAs revealed that expression of the TPO, thyroidstimulatmig hormone receptor (TSHR) and thyroglobulin (Tg) genes were athigh levels in thyroid gland, however, TSHR and Tg were detected in alsoseveral tissues including peripheral leukocytes. The TPO expression wasrestricted in thyroid and salivary gland, and its expression was not detected inperipheral leukocytes. Thirty-three thyroid papillary carcinoma patients of stageI (23 cases), II (7 cases) and III (3 cases), 69 non-cancer patients with benignthyroid diseases and 20 healthy volunteers were examined by RT-PCR for TPOand TSHR mRNAs using peripheral blood cells. The TPO mRNA in peripheralblood was detected in 61% cases of stage I thyroid papillary carcinomas, whileno case in 20 healthy volunteers. TSHR and Tg mRNAs were detected in 70%and 60% healthy volunteers, respectively, due to their expression in peripheralblood cells. Furthermore, we determined the number of thyroid follicular cells,thyrocytes, in circulation by using real-time monitoring quantitative RT-PCR.Cell number of thyrocytes in circulation was estimated in range from 1.1 to 2.7×103 cells/ml whole blood. Any relationship between cell number and stage,tumor size, or serum thyroglobulin level was not observed. Present fmdingssuggest that detection of tissue-specific mRNA in cancer like TPO mRNA inperipheral blood may be useful to diagnose early stage cancer as a potentialtumor marker.
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Kazuhiko Uchida, M. D.
Department of Biochemistry andMolecular OncologyInstitute of Basic Medical SciencesUniversity of Tsukuba1-1-1 Tennoudai, TsukubaIbaraki 305-8575, [email protected]
1983 Nara Medical University (M.D.)1983-1987 Department of Oncological Pathology
Nara Medical University (PhD, Medical Science)1985-1988 Research Resident, National Cancer Center Research Institute
(NCCRI), Tokyo1989 Research Staff, NCCRI1989-1995 Assistant Professor, Institute of Basic Medical Sciences,
University of Tsukuba, Tsukuba1995-present Associate Professor, Department of Biochemistry and
Molecular Oncology, Institute of Basic Medical Sciences,University of Tsukuba
1997- present Research Staff, Center for Tsukuba Advanced Research Alliance,University of Tsukuba
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List of Speakers and Chairpersons
Koichi Furukawa, M.D. ProfessorDepartment of Biochemistry IINagoya University School of Medicine65 Tsurumai, Showa-ku,Nagoya 466-0065, JapanPhone: 052-744-2070Fax: 052-744-2069E-mail: [email protected]
Sen-itiroh Hakomori, M.D. HeadDivision of Biomembrane Research,Pacific Northwest Research Institute,Seattle Washington, U.S.A.FAX: 206-726-1212E-mail: [email protected]
Gerald W. Hart, Ph.D. Professor & DirectorDepartment of Biological Chemistry,Johns Hopkins University School of Medicine,725 N. Wolfe St., Baltimore, MD 21205-2185Phone: 410-614-5993Fax: 410-614-8804E-mail: [email protected]
Nozomu Hiraiwa, M.D. Senior Research StaffLaboratory of Experimental PathologyAichi Cancer Center Research InstituteNagoya 464-8681, JapanPhone: 052-762-6111, ext. 8814Fax: 052-763-5233E-mail: [email protected]
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Masaru Ishii, M.D. President of Saitama Cancer CenterDivision of GastroenterologySaitama Cancer Center818 Komuro, Ina-Machi, Kitaadachi-Gun,Saitama, 362-0806, JapanPhone: 048-722-1111-ext. 4114Fax: 048-722-1129
Akiko Kanamori, Ph.D. Research StaffLaboratory of Experimental Pathology,Aichi Cancer Center Research Institute1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, JapanPhone: 052-762-6111, ext. 8815Fax: 052-763-5233E-mail: [email protected]
Reiji Kannagi, M.D. ChiefLaboratory of Experimental Pathology,Aichi Cancer Center Research Institute1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, JapanPhone: 052-762-6111, ext. 8812Fax: 052-763-5233E-mail: [email protected]
Geoffrey S. Kansas, Ph.D. Assistant ProfessorDepartment of Microbiology-Immunology,North-western Medical School,303 East Chicago Avenue, Chicago, IL 60611,Phone: 312-908-3237Fax: 312-503-1339E-mail: [email protected]
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Takashi Muramatsu, Ph. D. ProfessorDepartment of BiochemistryNagoya University School of Medicine65 Tsurumai-cho, Showa-kuNagoya 466-8550 JapanPhone: 052-744-2059Fax: 052-744-2065E-mail: [email protected]
Shoji Nakamori, M.D. Assistant ProfessorDepartment of Surgery II,Osaka University Medical School2-2 Yamadaoka, Suita,Osaka, 565-0871 JapanPhone: 06-879-3251Fax: 06-879-3259E-mail: [email protected]
Hayao Nakanishi, M. D. Section ChiefLaboratory of Pathology,Aichi Cancer Center Research Institute,1-1 Kanokoden, Chikusa-ku,Nagoya 464-8681, JapanPhone: 052-762-6111Fax: 052-763-5233E-mail: [email protected]
Hisanao Ohkura M.D. Vice DirectorIbaraki Regional Cancer CenterIbaraki Central HospitalTomobe-cho, Nishi-ibaraki-gunIbaraki 309-1793, JapanPhone: 0296-77-1121Fax: 0296-77-2886
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Makoto Ogawa, M.D. PresidentAichi Cancer Center1-1 Kanokoden, Chikusa-ku,Nagoya 464-8681, JapanPhone: 052-762-6111Fax: 052-763-5233
Suketami Tominaga, M.D. DirectorAichi Cancer Center Research Institute1-1 Kanokoden, Chikusa-ku,Nagoya 464-8681, JapanPhone: 052-762-6111Fax: 052-763-5233E-mail: [email protected]
Kazuhiko Uchida, M. D. Department of Biochemistry and MolecularOncologyInstitute of Basic Medical SciencesUniversity of Tsukuba1-1-1 Tennoudai, TsukubaIbaraki 305-8575, JapanPhone: 0298-53-3250Fax: 0298-53-3271E-mail: [email protected]
Ajit Varki, M.D. Professor of MedicineUCSD Cancer CenterUniversity of California San DiegoLa Jolla, CA 92093-0687Phone: 619-534-3296Fax: 619-534-5611E-mail: [email protected]
