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CANCERRESEARCH 56. 377-383.January 5. 19961 Predominant Expression of Human Zic in Cerebellar Granule Cell Lineage and Medulloblastoma' Naoki Yokota,2 Jun Aruga, and Katsuhiko Mikoshiba Setsuo Takai, Kiyomi Yamada, Minoru Hamazaki, Toshio Iwase, Haruhiko Sugimura, Molecular Neurohio!ogt' Laboratory, Tsukuba Life Science (‘enter, The Institute of Physical and Chemical Research (RIKEN.1, 3-1-I Koyadai, Tsukuba. Ibaraki 305 [N. Y.. J. A.. K. MI; Department of Microbiology and immunology (N. Y.J and 1st Department of Pathology (T. I., H. S.], Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, Shizuoka 431—31:Department of Pathology, Shizuoka Children ‘s Hospital. 860 Urushivama, Shizuoka 420 (Y. HI; Department of Genetics, Research institute, international Medical Center of Japan. 1-21-I Tovama-cho, Shinjuku-ku, Tokyo 162 (5. T., K. Y.J; and Department of Molecular Neurobiology, institute of Medical Science, The University of Tokyo. 4-6- 1 Shirokanedai, Minato-ku, Tokyo 108 (K. MI, Japan expressed in the granule cell lineage throughout the cerebellar devel ABSTRACT opment Zic is a novel zinc finger protein which displays a highly restricted expression pattern in the adult and developing mouse cerebellum and is highlyhomologous to the recentlyclonedDrosophilapair-rule geneOpa. To clarify the mechanismfor the developmentof the human cerebellum and its involvementin humannervoussystemdiseases, we haveisolated human Zic eDNA and examinedits expressionby using monoclonal antibodyagainstrecombinantZic protein.The nucleotidesequenceof humanZic cDNA is 85% homologousto that of mouseZic eDNA. Its putative amino acid sequenceis highly conserved (>99%) except for substitution of only two amino acid residues.In situ chromosomehybrid. in the mouse (9). Its zinc finger domain shows a high homology to the Drosophila pair-rule gene Opa (10), the Caeno rhabditis elegance sex determination gene tra-1 (1 1), the Drosophila segment polarity gene ci'@ (12), and the human oncogene Gil (13). Such similarities and expression patterns suggest that Zic plays a role in the developmentalprocess.Furthermore,Zic is potentiallyinvolved in the oncogenesisof undifferentiatednervoussystemtumors,includ ing medulloblastoma, since several oncogenes, such as Writ-I and GLI, exhibit expression in a spacially and temporally restricted pattern and play a role in the early organization of the structure (14, 15). izationlocalized thehumanZicgeneto chromosome band3q24.Human In the present study, to clarify the histogenesis of the human Zic protein was immunohistochemically detectedin the nuclei of the cerebellar granule cell lineage and its involvement in human nervous cerebellar granule cell lineage from the progenitor cells of the external system diseases,especially in medulloblastoma, we cloned human Zic germinallayer to the postmigratedcellsof the internal granular layer. cDNA and determined the chromosomal localization of the Zic gene Furthermore, Zic protein wasdetectedin medulloblastoma(26/29cases), by FISH. We also investigated its expression immunohistochemically whereas no other tumors examined (over 70 cases including primitive neuroectodermal tumors) expressed this protein. These findings in both normally suggest that Zic is a potentialbiomarker for medulloblastomaas well as the humancerebellargranulecell lineage. developing human cerebella and various pathologi cal specimens of human tumors. In addition, the diagnostic potential of immunohistochemical Zic protein in differentiating medulloblas toma from other tumors is discussed. INTRODUCTION MATERIALS The mammalian central nervous system contains an enormous number of neurons categorized into many subtypes. One of the major subjects of study is to understand the mechanism underlying the generation of these neuronal subtypes. Our interest has been in the processes of differentiation and maturation of cerebellar granule cells. Becauseof their unique maturation processesand mitotic ability in the postnatal period, we consider cerebellar granule cells an ideal system in which to analyze the neurobiological development. Another intriguing aspect of cerebellar granule cells is their in volvement in a certain type of brain tumor, medulloblastoma. Medul loblastoma, originally named by Bailey and Cushing (1), is a malig nant brain tumor, and accounts for approximately up to 25% of intracranial tumors in childhood (2). The origin of medulloblastoma has been ascribed to three main sources: fetal granular layers identical to the EGL3 (3), a “germinalbud―situated at the hind end of the posterior medullary velum (4), and the IGL (5—7). Medulloblastoma is categorized to cerebellar PNETs (8); however, the origin of this tumor is still controversial, and this category must be distinguished from other undifferentiated neuroepithelial tumors. We recently isolated a novel zinc finger protein, Zic, which is Received 8/2 1/95; accepted I 1/9195. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This study was supported in part by grants from the Institute of Physical and Chemical Research (RIKEN), Science and Technology Agency, Japanese Ministry of Education, Science, and Culture, and Japan Intractable Disease Foundation. 2 To I The whom requests abbreviations for used reprints are: should EGL, be extemal layer; IGL, internal eDNA and Genomic DNA Cloning. A cDNA libraryof humancerebellum in AgtI 1(ClontechLaboratories),whosemRNA sourcewasnormalcerebellar tissuefrom a 15-year-oldCaucasianfemale,wasscreenedat high stringency using 32P-labeledmouseZic cDNA containingalmostthe entirecoding region of Zic. Plaque lifts and subsequent purifications were performed essentially as describedby Sambrooket al. (16). Sequencingreactionsand nucleotide sequenceanalyseswereperformedaspreviouslydescribedby Arugaet a!. (9). The complete nucleotide sequence of three overlapping clones was determined on both strands. Using a human Zic cDNA clone as a probe, a genomic DNA library of Japaneseadult humansin EMBL3 provided by the the JapaneseCancer ResearchResourceBank(L1018)wasscreenedat high stringencyasdescribed above. The clones obtained were analyzed by restriction mapping and partial sequencing. Genomic Mapping. R-banded chromosomes were prepared using standard methods (18) with some modifications over 10 kb, which contains (19). The Zic genomic clone (pghzic8, almost the entire region of the human Zic gene) was used as a probe for FISH. The probe was labeled with biotin-16-dUTP (Boehringer) by nick translation. Hybridization was performed as reported (19, 20).A 10-foldexcessof totalhumanDNA wasaddedto thehybridization solution to suppressnonspecifichybridization. Signalswere detectedwith fluoresceinavidin DCS (Vector Laboratories).The slideswere stainedwith propidiumiodide(0.5 .&g/ml;Sigma)andexaminedundera Nikon Optiphot 2-EFD2 microscope (B2A filter). Kodak Ektachrome film (ASA 100) was used for microphotography of the chromosomes. Generationof mAb. The mouseglutathione-S-transferase—Zic fusionpro tein was isolated as described previously (9). BALB/c mice (6—8weeks old) addressed. germinal AND METHODS granular layer; FISH. fluorescencein situ hybridization;mAb, monoclonalantibody;GW, gesta tional week:PNET,primitive neuroectodermaltumor. wereimmunizedwith thefusionprotein.Themicereceivedaninitial injection of 100 i.@gprotein emulsified I:1 with Freund's complete adjuvant. Three weeks later, the mice were given a boost of 100 @tg protein in Freund's incomplete adjuvant. Two weeks after the boost, the serum samples were 377 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research. HUMANZic cDNACLONINGAND ITS EXPRESSIONIN VARIOU5TUMORS testedusinganELISA. Mice yieldinggoodserumresponses wereboostedwith 100 @xg proteinwith PBS3 daysbeforethe fusion.Spleencells werefusedto 5p2/O-Ag14myelomacells providedby Instituteof Physicaland Chemical Research (RIKEN) Cell Bank (RCB2O9) in the presence of PEG4000 (GLBCO), using standard protocols (21). Hybridoma supernatants were screened 1—2 weeks later with ELISA, immunohistochemistry, and immuno blot stripscontainingtheglutathione-S-transferase—Zic fusionprotein.Positive clones were cloned by limiting dilution. Ascites was produced as described human Zic as a homologue of mouse Zic cDNA and determined the entire nucleotide sequence of 3. 1 kb. The human Zic nucleotide sequence and deduced amino acid sequence are deposited in DNA Data Bank of Japan, European Molecular Biology Laboratory, and Genebank with the accession no. D76435. The human Zic nucleotide sequence and its putative open reading frame display 85 and 95% homology, respectively, with the mouse sequence. The open reading frame is 1344 bp (477 amino acids) long preceded by 786 nucleotides (21). The specificity of mAbs was checked by Western blot analyses. Immunohistochemistry. Formaldehyde-fixedparaffin-embeddedsections of an untranslated region and has a calculated molecular mass of 48.4 (3—6 @m) were preparedusing the usual procedures.The sectionswere kDa. Compared with the mouse Zic sequence,the human Zic-deduced dewaxed in xylene and hydrated in a series of ethanols. Since tissue antigens amino acid sequenceis highly conserved (>99%), with substitutions were markedlydecreasedin the tumor specimens,they were autoclavedin at only two amino acid residues,Ser―@ to proline, and Met422to 0.01% citrate buffer (pH 6.5) at 110°C for 10 mm as a meansof antigen leucine. In the zinc finger region sparing 227—384,the human Zic retrieval.The sectionswereincubatedwith 0.5%skim milk, 0.05%Tween20, 50 mMTris-HCI (pH 7.4),and 150mMNaC1for 60 mm at roomtemperature, amino acid sequence is identical to that of mouse Zic. Northern blot analysis indicates that the size of mRNA is 3.1 kb, followedby incubationwith anti-Zic mAb overnightat 4°C. Immunoreactivity which corresponds to the size of the cDNA. Whereas mouse Zic wasdetectedby an immunoperoxidasereactionusingthe materialsand pro ceduresprovidedin theVectastainkit (VectorLaboratories).3,3'-diaminoben mRNA consists of three different classes of cerebellar mRNA (2.9, zidinetetrahydrochloridewasusedasthe substrateof theperoxidasereaction. 3.2,and5.0kb), only oneclassof mRNA(3.1kb) wasdetectedin Zic Expressionin Developingand DevelopedHuman Cerebella.We humans (data not shown). This suggeststhat our cDNA clones cover examined immunohistochemically Zicexpression in pathological specimens of an almost full length of the mRNA. the normally developingcerebellaof eight prematureinfantsat GWs 19—37 The Human Zic Gene Was Mapped to 3q24. Southern blot and in the developedcerebellumof a 6-year-oldchild. analysis of human genomic DNA extracted from peripheral blood Zic Expressionin VariousHuman Tumors. Zic expression wasfurther mononuclear cells revealed that Zic is a single gene. A Zic genomic examined in various human tumor specimens obtained from 29 cases of clone (phzic8; see “Materials and Methods―)which contains almost medulloblastoma,I 1 casesof glioblastoma.10 casesof retinoblastoma,10 the entire human Zic gene (data not shown) was used as a probe for cases of ependymoma, 3 cases of Ewing sarcoma, 2 cases of choroid plexus papilloma,I caseof extracranialPNET,and 1caseeachof hemangioblastoma, FISH analysis. Forty-five of 50 (pro)metaphases observed showed ganglioneuroma, ganglioblastoma, malignant Schwannoma, alveolar soft part symmetrical double spots on at least one homologue of chromosome sarcoma,york sactumor,liposarcoma,immatureteratoma,giantcell sarcoma, 3. The signal region was localized to 3q24 (Fig. 1). No other chro andpineoblastoma. mosomes exhibited double spot signals. Thus, it was concluded that Zic expression was also looked for in a series of common carcinomas of epithelialorigin, such as gastric(six cases), colorectal(five cases), liver (four cases),breast(five cases),andlungcancer(six cases).Two casesof smallcell carcinomaandfour casesof carcinoidtumorswerealsoexamined. RESULTS The Human Zic SequenceIs Highly Conserved.We obtained three overlapping cDNA clones encompassing complete lengths of the human Zic gene is located at human chromosomal region 3q24. Several linkage analyses of degenerative cerebellar atrophies have been reported (23—28).However, none of the loci of the genes for degenerative nervous diseases, clarified by these linkage analyses, were identical to that of Zic. Zic Was Expressed in Human Premature Infantile and Ma hired Cerebella. A mAb, ZC26, could recognize Zic protein well using Western blot analyses and showed a good immunohistochemi 1@, 4@ ... 4 Fig. I . The human Zic localization to human chromosome 3q24 by FISH with R-banded chro mosomes. Arrows, spots of the signals after FISH using biotinated Zic genome DNA. ‘@@1 . 4 378 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research. @ @ -@I @‘ @ : ‘ @b, $.@ @•e@ HUMAN Zic cDNA CLONING AND ITS EXPRESSION IN VARIOUS TUMORS @ I:, @; ‘. @ i,-,- -‘ @ a @ @ .@. ‘p•• @@Ibqi, @ @ • • , S @ @ q •.. @ ,;@ . •4@ @ @ .. a•,p.@ g?@ @., • .@ . 4 • • • • •@.O ,@•_•@ . ‘.@ a •. •.• S 0 4. • . I • •• p • •‘/ ‘•*•‘4 .. ‘e @‘i S ‘z:'@ a • • a •..k• I.‘ ‘. • . 4•m, • 0•. ‘-#.@- @ . @ 0 I@ø, @ a. . 0 ) .., • aI •@s@' 40@ ‘,•r@ • S I? :@ .‘@ :@ • @ a.: @ a • •:, @ a • •• •..• SW. • • • a @l@••• a@ • . -@ ‘ •• •,@. • ,a.@ .@1 a •* d E@'@1@―@ C .A @ ,@w @@4ij,; .@ @-w;@; , a 0 • 0 0• @ .... a @.. S . ,‘ I _.•e @“ •: ‘0' ,. •@, .4. a @ •40 @ , @ ‘ ,v..@. , -p I • ••@ .@ •.@ • ‘ •0• @ @ 0 a. ‘,0@ •@#a S ‘ ‘q•: @ a.. . ••.s @ •@ •ø •,‘ @ •. I •..s @••@@0 v@ .. •@ .@ S : • *• •@• e r@Ii'S.I a a 4 0 • • ., •@ @‘ ‘@*, a . .a S a, • a 5, % @ . • @ a S I'- M “0 P•., •. .. @ .. .. ‘• •,•ta..@ ‘S @.. ‘@@t ., •@‘@ a:... “a. a @ ,.F@. @ . • :.,. 0• %@ a. ,ilr •@ • g “:•@ •1h@. . • Fig. 2. Immunohistochemical studies of Zic protein in human developing and developed cerebella. The cerebellum of a 21-OW premature infant: a, H&E stain. b, anti-Zic stain. The cerebellum of a 37-OW premature infant: c, H&E stain. d, anti-Zic stain. The cerebellum of a 6-year-old child: e, H&E stain. f, anti-Zic stain. Note that the nuclei of the cells in the EGL, inward-migrating granule cells, and postmigration cells in the IGL are strongly stained by anti-Zic immunohistochemical stain in 21 OW. The numbers of these inward-migrating cells decreased in 37 OW; however, anti-Zic-positive cells in the EGL were still persistent. In the developed cerebellum, the migration of granule cells is over, and the EGL has disappeared. Purkinje cells showed no reactivity against anti-Zic antibody in any stages.E, external germinal layer. P. Purkinje cell; G, internal granular layer; M. molecular layer. X80. Bar, 100 @m. 379 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research. HUMAN Zic cDNA CLONING AND ITS EXPRESSION Table I hnmunohistochemistr, of anti-Zic mAb in various human tumors in Table Brain tumors Medulloblastoma Extracranial tumors Ewing sarcoma PNET 26/29 0/3 0/I Other intracranialtumors 0/10 0/I 0/2 0/10 0/I I 0/3 0/I Retinoblastoma Pineoblastoma Choroid plexus papilloma Ependymoma(ependymoblastoma) Glioma (glioblastoma) Ganglioneuroma (ganglioblastoma) Hemangioblastoma Other cancers Gastriccancer a Number of Zic-positive cases/number 0/4 0/I 0/I 0/I 0/I 0/1 0/1 0/I of examined is a phylogenically cases. cal reactivity. It was also revealed that the mAb cross-reacted with other members of the Zic family because of high homology of their carboxy terminus.4 However, their expression patterns were almost the same as that of Zic protein, and the amount of expression was quite low according to Northern blot analysesand in situ hybridization studies. Furthermore, the immunohistochemical localization of Zic protein in rat central nervous system using this mAb correlated well with the results of previous in situ hybridization studies in mice and immunohistochemical studies using the polyclonal antibody5 (9). Thus, we used this mAb in the subsequent immunohistochemical analyses. A four hundred-fold dilution of ZC26 appeared to result in distinctive nuclear staining without background. Zic protein was strongly detected in the nuclei of EGL component cells and in those of inward-migrating granule cells and postmigrated granule cells in the IGL, both in the developing and developed cerebella (Fig. 2). These results indicate that human Zic protein is predominantly expressed in the nuclei of the cerebellar granule cell lineage throughout development. Expression of Zic Protein in Human Tumors. Table 1 summa rizes the profile of the results of immunostaining in various human tumors, and we reviewed all cases of medulloblastoma, Ewing sar coma, and PNET concerning clinical information such as age, sex, and location in Table 2. Immunohistochemical positiveness of Zic protein was highly restricted to cases of medulloblastoma. A fraction in the cells with round nuclei and fine chromatin structure, whereas the component of pyknotic tumor cells and the cells with neuronal or glial differentiation were weak or negative (Fig. 3). Three casesof medulloblastoma (cases7, 9, and 11 4 .1. Aruga, T. Nagai, T. Tokuyama, Y. Hayashizaki, Y. Okazaki, V. M. immunoreactivity was Chapman, gene that may participate in an indis Medulloblastoma caseAgeSexLocation1 yr 2 3 4 10 5 yr M M M Vermis Vermis Vermis 10 yr F Vermis 6 7 8 9 10 11 1 11 9 4 9 8 yr 8mo yr 4mo mo yrllmo mo yrlOmo 12 6 yr M M M M M F M M F F F M F M M F M M F M F F F FVermis Vermis Vermis Vermis Vermis Hemisphere Vermis Vermis Vermis Posterior fossa Vermis Hemisphere Vermis Vermis Vermis Vermis Vermis Vermis Vermis Vermis Hemisphere Hemisphere Hemisphere Vermis yr yr yrF F MThigh Rib Pelvis yr M Submandibular region 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 HemisphereEwing 298 sarcoma I 2 316 and odd-paired, manuscript submitted for publication. N. Yokota, J. Aruga. and K. Mikoshiba. Immunohistochemical localization of Zic protein in the central nervous system of developing and adult rat, manuscript in prepa ration. conserved Table 2 Clinical profiles of medulloblastoma and PNET K. Mikoshiba.ThemouseZicgenefamily:homologuesof theDrosophilapairrulegene @ The pensable function. Its genomic organization is also highly homolo gous to that of the mouse.4 Recently, the Drosophila pair-rule gene Opa, which is highly homologous (over 70%) to Zic in the zinc finger region, was isolated (10), suggesting that Zic is a mammalian homologue of Opa.4 Opa is required for timely activation of wingless and engrailed in the para segment subdivision of the Drosophila embryo (10). Both Wnt-1 and En-2, which are mammalian homologues of wingless and engrailed, respectively, are involved in cerebellar development according to the of medulloblastoma cells in the majority of the cases tested showed positive immunoreactivity of anti-Zic mAb in the nucleus, while there appeared to be no immunoreactivity in other tumors (Table I). In medulloblastoma cells, the positive immunoreactivity of anti-Zic mAb was present predominantly for Zic protein. Human Zic Is Highly Homologous to the Mouse Zic Gene. We isolated cDNA and genomic clones of human Zic. The deduced amino acid sequence is highly homologous to that of the mouse (99.6%), and the homology is significantly higher than that in other developmen tally regulated genes,such as that between human GLI and mouse GLJ (88.4%; Ref. 15) and that between human En2 and mouse En2 (90%; Ref. 29). Our previous Southern blot analyses showed that there are homologues of mouse Zic in various vertebrates (carp, frogs, lizards, chicks, and humans; data not shown). These findings suggest that Zic Miscellaneoustumors Carcinoid tumors Malignant lymphoma Immature teratoma Malignant Schwannoma Alveolar soft part sarcoma Yolk sac tumor Liposarcoma Giant cell sarcoma negative DISCUSSION 0/6 0/5 0/4 0/6 0/5 Colorectal cancer Livercancer Lung cancer Breast cancer 2) were unrelated to clinicopathological features such as location, age, and stages, or no particular histopathological characteristics were identi fled in these cases. Ewing sarcoma and PNETs in s.c. region also exhibited negative reactivity, although histological photographs re vealed that they were small round cell tumors indistinguishable from medulloblastoma. Zic protein was unambiguously absent in the rather common epithelial neoplasms and common brain tumors such as glioma(Fig.3). Casea Histological diagnosis IN VARIOUS TUMORS 1 yr 8mo 4 yr 8 yr 3mo 4 yr 3 yr 13 yr 2 yr Smo 1 yr 6mo 1 yr 4mo 6 yr 3mo 5 yr 3 yr 4 yr 2 yr 6 yr 2 1 yr 48 yr 35 yr 40 yrM― 10 15 PNET S a M, male; F, female. 380 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research. HUMAN Zic cDNA CLONING AND ITS EXPRESSION IN v@aious TUMORS d :a @ S •@ @ ‘@a:@ :@ •., . . ‘* . V $@ @:“-@‘@ Fig. 3. lmmunohistochemical studies of Zic protein in a medulloblastoma and a glioblastoma. a, a 3-year-old male with classical-type medulloblastoma of the cerebellar vermis. H&E stain.b, anti-Zicimmunohistochemical stainof thesamecase.Thenucleioftumor cellswith roundnucleiandfine chromatinstructurearestronglystainedin medulloblastomas, whereas thecytosolof cellsandadjacent structures fail toexpress immunoreactive Zicprotein.c,glioblastoma multiforme of thecerebral hemisphere in a middle-aged female,whichshows typical histological features such as pseudopallisading, necrosis, and endothelial proliferation. Photomicrograph displays hypercellular and relative anaplastic parts. H&E stain. d. anti-Zic immunohistochemical stain in the same case. Note that tumor cells are unambiguously negative in immunoreactivity of Zic protein. X200. Bar, 20 p.m. results of gene-targeting studies (30—32). Although it is uncertain whether the hierarchy in Drosophila is conserved in the mammalian cerebellum, Zic may play important roles in the development of cerebellum. Zic Expressionin Normal HumanCerebellum.In humans,the EGL is formed by a number of cells which migrate through the marginal layer from the neuroepithelium to the surface of cerebellum in the 12-week embryo. In the 6th month, the EGL begins to release granule cells which migrate inward and pass through the differenti ating Purkinje cell layer to form the IGL. The production and migra tion of granule cells continues for 1.5 years after birth (33). In our study, Zic protein was expressed in the nuclei of the EGL component cells and in those of inward-migrating granule cells and postmigrated granule cells in the IGL, both in the developing and developed cerebella. The immunostaining in this study supports an idea that Zic is persistently expressed in the granule cells and its precursor cells in various species. The results of this study also suggest that Zic will be useful in pathophysiologically detecting the depopulation of granule cells caused by viral infection and toxicosis at the fetal stage. Predominant Expression of Zk in a Distinct Type of Human Tumor, Medulloblastoma. We examined various human tumors im munohistochemically and demonstrated a highly restricted expression of Zic protein in medulloblastoma, whereas no specific antigen has thus far been reported as a marker of medulloblastoma. As previously stated, our studies show that Zic is expressed abundantly but restrict edly in the cerebellar granule cell lineage. Although it is uncertain that medulloblastoma are committed to a course of the cerebellar granular cell lineage, our observations support the idea that medulloblastoma exhibits a differentiation characteristic of the cerebellar granule cell lineage in terms of expression of Zic, and this particular population of the cells probably represents the origin of medulloblastoma. Some immunohistochemicalstudies have demonstratedthe predominant capacity of medulloblastoma cells to differentiate along neuronal cell lines (34—37)and have shown that medulloblastoma shares the same immunological features as postmitotic cells forming the EGL of the cerebellum (38—41).These findings are also compatible with the results of our immunohistochemical analyses demonstrating the pres ence of Zic protein in medulloblastoma. 381 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research. HUMAN Zic cDNA CLONING AND ITS EXPRESSION IN VARIOUS TUMORS Concerning the histogenesis of medulloblastoma, there has been an argument about whether the entity of medulloblastoma is unique in cerebellar tumor or a more ubiquitous tumor derived from younger neuroblasts distributed throughout the central nervous system (36, 42—47).The recent concept on the tumor entity of the PNET has complicated the issue. According to the new edition of the WHO book on “Histological Typing of the Central Nervous System,― the diag nosis of PNET is recommended only as a generic term for cerebellar medulloblastoma and neoplasms that are histologically indistinguish able from medulloblastoma but located in the central nervous system at sites other than the cerebellum (8). However, although PNETs, regardlessof their locations, appearto be histologically identical, their biological behavior may vary considerably with location; patients with supratentorial PNETs have much worse prognoses than patients with medulloblastoma, despite similar treatments (17). Furthermore, it was shown that medulloblastoma has a different cytogenetical profile from other PNETs (22). Our findings suggest that medulloblastoma differs from other PNETs or Ewing sarcoma of a more undifferenti ated origin in terms of expression of Zic. It is possible that medullo blastoma should be classified into a distinct tumor entity which would differentiate it from other PNETs. Since few biomarkers have been known for medulloblastoma, the potentiality of this protein is highly expected for differential diagnosis of medulloblastoma from other brain tumors, especially undifferentiated neuroectodermal tumors. Further characterization of the variety of human tumors, especially central and peripheral PNETs, by immunohistochemical analyses using the anti-Zic mAb are needed to establish its diagnostic signif icance. ACKNOWLEDGMENTS We thankH. Igarashi(1stDepartmentof Pathology,HamamatsuUniversity Schoolof Medicine)for technicalassistance;M. Fukuda,M. Hashimoto,and other members of Mikoshiba's laboratory for valuable discussions; Dr. A. Miyawaki (Institute ing adult cerebellar of Medical Science, The University tissues; Dr. K. Sato (University of Tokyo) for provid of Okayama) for patho I I. Zarkower, D., and Hodgkin, J. Molecular analysis of C. elegance sex-determining gene tra-l: a gene encoding two zinc finger proteins. Cell, 70: 237—249,1992. 12. Orenic, T. V., Slusarski, D. C., Kroll, K. L., and Holmgren, R. A. Cloning and characterization of the segment polarity gene cubitus interruptus Dominant of Drosophila. Genes & Dcv., 4: 1053—1067,1990. 13. Kinzler, K. W., Ruppert, J. M., Bigner, S. H., and Vogelstein, B. The GU gene is a memberof theKruppelfamily of zinc finger proteins.Nature(Lond.),332: 371—374, 1988. 14. Roelink, H., and Nusse, R. Expression of two members of the Wnt family during mouse development—restrictedtemporal and spacial pattern in the developing neural tube. Genes & Dcv., 5: 381—388,1991. 15. Waterhouse, D., Ahmed, M., Slusarski, D., Kalamaras, J., Boucher, D., Hormgren, R., and lannaccone, P. gli, a zinc finger transcription factor and oncogene. is expressed during normalmousedevelopment.Dcv. Dyn., 196: 91—102, 1993. 16. Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning, pp.2.60—2.80.Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989. 17. Rutka, J. T., and Hoffman, H. J. A critical review of medulloblastoma; from a difficult past to a promising future. Neurosurg. Quart., 1: 54—78,1991. 18. Viegas-Pequignot, E., and Dutrillaux, B. Une Methode simple pour obtenir de sprophaseset des prometaphases. Ann. 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Neurol.,42: 1—15, 1984. 383 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research. Predominant Expression of Human Zic in Cerebellar Granule Cell Lineage and Medulloblastoma Naoki Yokota, Jun Aruga, Setsuo Takai, et al. Cancer Res 1996;56:377-383. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/56/2/377 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research.