BRIEF REPORT |
Correspondence to: Andy J.G. Pötgens, Dept. of Anatomy, University Hospital Aachen, Wendlingweg 2, D-52057 Aachen, Germany. E-mail: apotgens@ukaachen.de
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Summary |
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CD133 is an antigen expressed on hematopoietic progenitor cells and on some epithelial cells. We previously reported that a commercially available antibody against CD133, CD133-2/AC141, also reacted with an intracellular protein in placental trophoblasts. Here we show by 2D electrophoresis and mass spectroscopy that this reactivity is with cytokeratin 18, a cytokeratin present in most simple epithelia. Immunohistochemistry (IHC) with CD133-2/AC141 on a trophoblast cell line displayed a staining pattern typical for the cytoskeleton. Cryostat sections of stratified epithelia lacking cytokeratin 18 did not react with CD133-2/AC141. In conclusion, care must be taken not to misinterpret staining patterns using CD133-2/AC141 in IHC. (J Histochem Cytochem 50:11311134, 2002)
Key Words: CD133, AC141, PROML1, cytokeratin 18, hematopoietic stem cells, epithelial cells, human
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Introduction |
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CD133 is a cell surface glycoprotein localized in membrane protrusions or microvilli. It is expressed by stem and progenitor cells developing into hematopoietic lineages (
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We tried to identify the protein responsible for this reactivity. Cell extracts were prepared from a choriocarcinomatrophoblast hybrid cell line, AC-1M32 (
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Cytokeratin 18 is a member of the family of acidic cytokeratins (cytokeratins 920) that join with basic cytokeratins (18) to produce the intermediate filaments of epithelial cells. Cytokeratin 18 is expressed mainly in simple epithelia, combines with cytokeratin 8 (
IHC confirmed reactivity of AC141 with a cytoskeletal protein in trophoblast cell line AC-1M32. Cells cultured on eight-chamber glass slides were fixed for 10 min in acetone at -20C, dried, rehydrated in PBS, pretreated in methanol/hydrogen peroxide, and immunostained with AC141 essentially as in
To further confirm that cytokeratin 18 is the AC141-reactive protein in trophoblast, we incubated 2D blots with an MAb against cytokeratin 18 (clone Cy90, Sigma, Taufkirchen, Germany; diluted 1:10,000), rabbit anti-mouse, and PAP, as above, and developed the blot in AEC substrate for 5 min (Fig 1). Anti-cytokeratin 18 incubation revealed a row of spots of 48 kD having a pI of 5.55.9, as well as some weaker spots of about 52 kD. The staining pattern after incubation with anti- cytokeratin 18 was very similar to that seen after AC141 staining. According to the literature (
Crossreactivity of AC141 with other cytokeratins than cytokeratin 18 cannot be excluded. Trophoblasts and other simple epithelia express only a subset of all cytokeratins, including cytokeratin 18. Other sets of cytokeratins are found in stratified epithelia, such as cytokeratins 1, 2, and 10 (typical for keratinized skin-type epithelia) or 4 and 13 (typical for unkeratinized stratified squamous epithelia) (
To study reactivity of AC141 with various types of epithelia, we immunostained acetone-fixed cryostat sections of placenta (obtained after spontaneous birth), ilium, gingiva, axillar skin (all three removed for diagnostic purposes during surgery), and foreskin of penis (removed by circumcision) with AC141 (diluted 1:50; method as above). Placental trophoblast, mucosal epithelium of ilium, and eccrine sweat glands, all of which are simple epithelia, reacted with AC141 (Fig 2B2D). Keratinized and unkeratinized stratified epithelia, such as those in hair follicle, epidermis, and gingiva, did not show reactivity with AC141 (Fig 2D2F). The stratified epithelia did react, however with a pan-cytokeratin antibody (MNF116, DAKO; diluted 1:300), as shown in Fig 2G and Fig 2H. AC141 therefore reacted with various epithelia expressing cytokeratin 18 but not with epithelia lacking cytokeratin 18.
In conclusion, an MAb, AC141, raised against membrane antigen CD133, displays additional reactivity with cytokeratin 18, a cytoskeletal protein present in many epithelia. Although some epithelial cells actually do express CD133 (
AC141 can be used as an alternative antibody against cytokeratin 18 in IHC. It should be noted, however, that it displays this reactivity only in cryostat sections, not in paraffin sections (
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Acknowledgments |
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Supported by funds from the University of Technology Aachen (START 78/2000 to AP), the Deutsche Forschungsgemeinschaft (DFG, grants no. FR1245/3-2,3 to HGF and PK), and the Rockefeller Foundation (grants no. RF 96020#76, RF98024#99, RF99021#114 to PK and HGF).
We thank Angela Rüben and Anke Gärtner for excellent technical assistance and Drs Frank Jugert, Thorsten Laurin, Dennis von Heimburg, and Sören Gärtner (University Hospital Aachen) for their generous gifts of tissue specimens.
Received for publication December 12, 2001; accepted April 3, 2002.
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Literature Cited |
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Cooper D, Schermer A, Sun T-T (1985) Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: strategies, applications, and limitations. Lab Invest 52:243-256[Medline]
Corbeil D, Röper K, Hellwig A, Tavian M, Miraglia S, Watt SM, Simmons PJ et al. (2000) The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions. J Biol Chem 275:5512-5520
Frank H-G, Gunawan B, EbelingStark I, Schulten HJ, Funayama H, Cremer U, Huppertz B et al. (2000) Cytogenetic and DNA-fingerprint characterization of choriocarcinoma cell lines and a trophoblast/choriocarcinoma cell hybrid. Cancer Genet Cytogenet 116:16-22[Medline]
Haigh T, Chen C-P, Jones CJP, Aplin JD (1999) Studies of mesenchymal cells from 1st trimester human placenta: expression of cytokeratin outside the trophoblast lineage. Placenta 20:615-625[Medline]
Heukeshoven J, Dernick R (1988) Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels. Electrophoresis 9:28-32[Medline]
Johansen M, Redman CWG, Wilkins T, Sargent IL (1999) Trophoblast deportation in human pregnancyits relevance for pre-eclampsia. Placenta 20:531-539[Medline]
Moll R, Franke WW, Schiller DL (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11-24[Medline]
Mühlhauser J, Crescimanno C, Kasper M, Zaccheo D, Castellucci M (1995) Differentiation of human trophoblast populations involves alterations in cytokeratin patterns. J Histochem Cytochem 43:579-589
Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane WJ, Williams M, Oz MC et al. (2000) Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors.. Blood 95:952-958
Pötgens AJG, Bolte M, Huppertz B, Kaufmann P, Frank H-G (2001) Human trophoblast contains an intracellular protein reactive with an antibody against CD133a novel marker for trophoblast. Placenta 22:639-645[Medline]
Uchida N, Buck DW, He D, Reitsma MJ, Masek M, Phan TV, Tsukamoto AS et al. (2000) Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci USA 97:14720-14725
Yin AH, Miraglia S, Zanjani ED, AlmeidaPorada G, Ogawa M, Leary AG, Olweus J et al. (1997) AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 90:5002-5012