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Journal of Cell Science 108, 97-103 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

Identification of the cells expressing cot proto-oncogene mRNA

Reiko Ohara1,*, Seiichi Hirota2, Hitoshi Onoue2,†, Shintaro Nomura2, Yukihiko Kitamura2

and Kumao Toyoshima1,‡

1Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita 565,Japan2Department of Pathology, Osaka University Medical School, Yamadaoka 2-2, Suita 565, Japan

*Author for correspondence at present address: Laboratory of DNA Technology, Kazusa DNA Research Institute, Yana-uchino 1532-3, Kisarazu 292, Japan†Present address: Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235, USA‡Present address: The Center for Adult Diseases, Osaka, Higashinari-ku Nakamichi 1-3-3, Osaka 537, Japan

The cell types expressing cot proto-oncogene mRNA wereidentified by in situ hybridization (ISH) histochemistry.Among a variety of adult mouse tissues examined, fourtypes of glandular cells expressing cot gene were identified:(1) granular duct cells in the submandibular and sublin-gual glands; (2) serous cells in the parotid gland; (3) peptic(chief) cells in gastric glands; and (4) goblet cells in colonicglands. Investigation of the developmentally regulatedexpression of cot mRNA using tissues of 14-day and 18-dayembryos, newborn and weanling mice showed that cot geneis expressed only in morphologically differentiated andfunctionally activated cells of these four types. No othertypes of cells showing ISH signals were observed. Based on

these results, cot gene expressions in cultured cells ofcolonic adenocarcinomas and gastric adenocarcinomaswere examined. SW 480 and WiDr cells showed highexpression of this gene and so should be useful for func-tional analysis of Cot kinase. The expression patterns of cotgene in tumor tissues of the parotid gland, and gastric andcolonic glands were investigated. Two of the tissues over-expressed this gene markedly, suggesting that overproduc-tion of Cot kinase may be one cause of their transforma-tion.

Key words: cot proto-oncogene, in situ hybridization, glandular cell

SUMMARY

INTRODUCTION

Cells are known to have various signal transmission pathwaysby which many extracellular stimuli are transmitted from themembrane to the nucleus during the processes of growth anddifferentiation. Many proto-oncogene products take part inthese pathways (Cantley et al., 1991), so their activation bymolecular aberration inevitably causes overgrowth and/ordeviated cell differentiation, and leads to transformation (Joveand Hanafusa, 1987; Reddy et al., 1982; Downward et al.,1984; Lewin, 1991).

Of the proto-oncogenes encoding serine/threonine kinase,the mos (Watson et al., 1982; Schmidt et al., 1988), raf (Bonneret al., 1985, 1986), pim (Selton et al., 1986; Padma andNagarajan, 1991), cot (est) (Miyoshi et al., 1991; Aoki et al.,1991, 1993; Chan et al., 1993) and atk (rac) (Bellacosa et al.,1991; Jones et al., 1991) genes have so far been cloned. Inter-estingly, as they each possess unique structures (Hanks andQuinn, 1991), they are supposed to have distinct functions, butonly those of Mos (Sagata et al., 1988, 1989) and Raf (Dent etal., 1992; Howe et al., 1992; Kariakis et al., 1992) kinases haveyet been clarified. On the other hand, a signal transductioncascade mediated by serine/threonine kinases was recentlyreported: Raf-1 kinase, MAP (mitogen-activated protein)

kinase kinase kinase, MAP kinase kinase, MAP kinase andribosomal S6 kinase function in this pathway and contribute tosignal transmission by consecutive phosphorylation (Dent etal., 1992; Howe et al., 1992; Kariakis at al., 1992; Pelech andSanghera, 1992; Seger et al., 1992; Blenis, 1993; Lange-Carteret al., 1993; Wu et al., 1993). Furthermore, some counterpartsof these kinases were found in yeasts (Teague et al., 1986;Nadin-Davis and Nasim, 1988; Courchense et al., 1989; Elionet al., 1990; Rhodes et al., 1990; Toda et al., 1991; Wang etal., 1991), Drosophila (Biggs and Ziprusky, 1991; Tsuda et al.,1993) and Xenopus (Kosako et al., 1992, 1993; Matsuda et al.,1992). Thus this cascade seems to be conserved from lowerorganisms to mammals. Accordingly, it would be interestingto know how other proto-oncogenes encoding serine/threoninekinases are involved in this or other signal transductionpathways, and which roles they have in cell growth and differ-entiation.

The cot oncogene was first identified as a rearranged formduring transfection assays with SHOK cells (Miyoshi et al.,1991). Since then, its prototype has been cloned, and analyseswith antibodies have been performed. Results have shown that:(1) this kinase is localized in the cytoplasm; (2) two different-sized Cot kinases of 58 kDa and 52 kDa protein are producedby an alternative initiation mechanism and have different trans-

98 R. Ohara and others

Fig. 1. ISH results on adult tissues. (A, B) Submandibular gland. (A) The result of ISH.After counterstaining with saffranin, hybridized signalsappeared purple but nuclei red. Signals were detected ongranular ducts but not on striated ducts (*). Byobservation of a HE-stained section (B), the cell typescould easily be distinguished (see text). Gd, granularduct; Sd, striated duct; Ma, mucus acinus. (C, D) Sublingual gland. Hybridized signals were seenas dots (C). HE staining of a serial section (D)demonstrated that some cells similar to granular ductcells constituted striated ducts (indicated by arrowheads)and they gave positive signals. (E, F) Parotid gland. Theresult of ISH (E) and a serial section stained with HE (F)

are shown. Signals were observed on the serous acinar cells (Sa), which are predominant in the this gland. Striated ducts showed no signal, asin the submandibular gland. (G, H) Gastric glands. The cells that hybridized with cot probe were located in the basal area of the stomach (G).By comparison with a serial section stained with HE (H), cot-positive cells were identified as peptic (chief) cells (Pc), which have a basophiliccytoplasm. The other type of cells, parietal cells, which have an eosinophilic cytoplasm, did not react with cot probe (arrowheads). (I, J) Colonic glands. Sections were counterstained with haematoxylin, and nuclei are blue. A longitudinal section (I) and a transverse section(J) are shown. Both figures showed signals on younger goblet cells located in the basal area of the mucosa, but not on older goblet cells (largearrowheads) or adsorptive cells (small arrowheads). Bars, 5

µm.

forming activities; (3) rearrangement of the carboxy-terminalregion of this kinase increases its transforming activity (Aokiet al., 1993); and (4) in a murine system, this gene is tran-scribed in many tissues from the embryo to adult stage,although its level differs from tissue to tissue (Ohara et al.,1993). However, the physiological function of Cot kinase isnot yet known, mainly because its expression level is in general

low and no appropriate cell system for its biochemical analysishas yet been established.

In this work, to overcome this problem, we tried todetermine the cell types expressing cot mRNA by ISH histo-chemistry. Results showed that four types of glandular cellshybridized with cot probe, and that Cot protein may beinvolved in secretory phenomena.

99Cells expressing

cot mRNA

Fig. 2. ISH results on tissues duringdevelopment. (A, B) Submandibulargland at the weaning stage. The result ofISH is shown in (A), and a serial sectionstained with HE in (B). Acinar formationis already complete, but granular ducts arenot yet differentiated. Although striatedducts (Sd), from which granular ducts areknown to differentiate, were observed, nosignal was detected on them. Sa, serousacinus; Ma, mucus acinus. (C, D) Parotidgland at weaning stage. Morphologically,the tissue is as well differentiated as in anadult mouse (D). The result of ISH wasthe same as in adult tissue, positivesignals being detected on serous acini, butnot on striated ducts (C). (E, F) Gastricgland of a weanling mouse. The tissue ofthe gastric gland appeared as welldeveloped as that in an adult mouse (F).ISH signals were detected on peptic cells,but not on parietal cells (arrowheads) (E).(G, H) Colonic glands of a newbornmouse. Goblet cells have differentiated,but give no ISH signal (G and H are notserial sections but close together). (I) Colonic glands at the weaning stage.The section was counterstained withhaematoxylin after ISH. The mucosallayer is better developed than in newbornmice, and an ISH signal was detected ongoblet cells in the basal area. Bars, 5 µm.

MATERIALS AND METHODS

Animals, tissue preparations and in situ hybridization(ISH)Balb/c and C57 B6 strain mice were purchased from Shizuoka Lab-oratory Animal Center (Hamamatsu, Japan). Embryos (14-days and

18-days) and newborn mice were fixed whole, whereas various organswere excised from weaning and adult mice for fixation. After fixationwith 4% paraformaldehyde (PFA) in PBS (pH 7.4), the preparationswere dehydrated and embedded in paraffin wax. Sections were trans-ferred to 3-aminopropyltriethoxysilane-coated slides and stored at4°C until use. The protocols of Nomura et al. (1988) and Hirota et al.

100 R. Ohara and others

(1992) were used for pretreatment and hybridization. The murine cotcRNA probes used were transcribed in vitro with a DIG RNALabeling Kit (Boehringer Mannheim, Germany) according to themanufacturer’s instructions. We used two non-overlapping probescorresponding to 383 bp SpeI (probe no. 5) and 1,147 bp Pst I (probeno. 6) fragments of the CZ-21 clone (Ohara et al., 1993). The cDNAsof these PstI and SpeI fragments were subcloned into pBluescript IISK(−) vectors. For obtaining the anti-sense probe no. 5, the plasmidwas linearized with XbaI and transcribed with T7 RNA polymerase,whereas for the sense probe, the plasmid was linearized with BamHIand transcribed with T3 RNA polymerase. Anti-sense probe no. 6 wasobtained by digesting the plasmid with EcoRI and transcription withT3 RNA polymerase, whereas the sense probe was obtained bydigestion with BamHI and transcription with T7 RNA polymerase.After hybridization, the sections were washed and signals weredetected as described (Hirota et al., 1992). The cot-positive cells wereidentified as those giving signals with both anti-sense probes no. 5and no. 6, but not with sense ones. Counterstaining for identificationof the nucleus was performed using safranin or haematoxylin dye, andfor identification of cell types, serial sections were stained withhaematoxylin-eosin (HE). The development and tissue histology ofmice were examined with reference to the books by Kaufman (1992),Sreebny (1987) and Wheater et al. (1987).

Cultured cell linesAZ 521, CoLo 201, CoLo 320 DM and MKN 45 cells were suppliedby the Japanese Cancer Research Resources Bank (JCRB) (Tokyo),Lovo, LS 180, SW 480 and WiDr cells were from the American TypeCulture Collection (ATCC), and KATO III cells were from Dr Seiki(Cancer Research Institute, Kanazawa University).

Human tissuesHuman tissues of patients 1, 2 and 3 were obtained from the OsakaUniversity Medical Hospital and those of patients 4, 5 and 6 werefrom the Attached Hospital of the Research Institute of MicrobialDiseases (Osaka University).

Northern blot analysisCellular RNAs were purified by the acid-guanidinium-phenol-chloro-form (AGPC) method (Chomczynski and Sacchi, 1987). For mRNApurification, Oligo-Tex dT(30) super (Japan Synthetic Rubber Co.,Ltd.) was used following the manufacturer’s instruction. Samples of1 µg of mRNA (of cultured cells) or 30 µg of cellular RNA (of tissues)were fractionated in 1% formaldehyde agarose gel and transferred tonylon membranes. Hybridization and washing were performed asdescribed before (Ohara et al., 1993). The probe used was a 2.3 kbEcoRI fragment of human cot cDNA (Aoki et al., 1993), which waslabeled by the random priming method. As a control, membranes werewashed and rehybridized with human glyceraldehyde 3-phosphatedehydrogenase (G3PDH) cDNA (Clontech, USA). The membraneswere exposed to Kodak O-mat film at −70°C for 3 days and 10 daysto detect cot gene expression in human cell lines and tissues, respec-tively, but for 1 day to detect G3PDH.

RESULTS

ISH on adult mouse tissuesPrevious studies by northern blot analysis showed that manytissues express cot mRNA, but that its level differs from tissueto tissue. Here, to identify the cell types that express cotmRNA, we carried out ISH histochemical analyses using cotcRNA probe. On examination of the salivary glands (sub-mandibular, parotid and sublingual glands), thymus, lung,liver, spleen, stomach, colon and ovary, positive signals were

detected in four types of glandular cells: granular duct cells inthe submandibular and sublingual glands, serous cells in theparotid gland, peptic cells in gastric glands and goblet cells incolonic glands.

The results of ISH of submandibular gland tissue are shownin Fig. 1A. The pattern of positive signals was consistent withthe ductal configuration. By observation of an HE-stainedsection (Fig. 1B), the cells giving ISH signals were identifiedas those of granular ducts, which develop in rodents afterpuberty and produce proteases and hormone-like substances.Cells of granular ducts have nuclei located in the basal areaand many granules in the cytoplasm, and so can be distin-guished from those in striated ducts, which have basal stria-tions and nuclei located in the upper region of the cell, andwhich gave a negative reaction with cot probe (indicated byasterisks in Fig. 1A).

In the sublingual gland, hybridization signals appeared to belocalized as dots (Fig. 1C). To identify the cells that hybridizedwith cot probe, we stained a serial section with HE, as shownin Fig. 1D. On comparison of the two sections, we noticed thatsome cells that were very similar to granular duct cells werepresent in the striated ducts (indicated by arrowheads in Fig.1D), and gave positive signals.

In the parotid gland, hybridized signals were observed inmost of the tissue (Fig. 1E). An HE-stained serial sectiondemonstrated that serous acini, which are known to secreteamylase, maltase and ptyalin, hybridized with the cot probe,but striated ducts did not (Fig. 1F).

We also detected cot mRNA expression in gastric glands.The signals were detected around the basal area of the stomachbody, as shown in Fig. 1G. Since HE-staining of a serialsection revealed that these signals were on cells possessinghighly basophilic cytoplasm and a basally located nucleus (Fig.1H), we concluded that they were peptic (chief) cells knownto produce the pepsin precursor pepsinogen. Another type ofcells, parietal cells, also present in the basal area of the mucosa(arrowheads in Fig. 1H), did not hybridize with cot probe.

Results on colonic glands are shown in Fig. 1I (longitudinalsection) and J (transverse section). There are two types of cellsin the mucosal layer: mucus-secreting goblet cells and water-recovering adsorptive cells. From these figures, it is clear thatISH-positive signals were observed only around the base of themucosal layer where younger goblet cells are lined up bycontinual mitoses. No signal was detected on older goblet cells(large arrowheads) or adsorptive cells (small arrowheads),which are located in the middle and upper regions of themucosa.

ISH during mouse developmentNext, to investigate the dependence of cot mRNA expressionon the developmental stage, we carried out ISH on paraffinsections prepared from 14-day and 18-day embryos, andnewborn and weanling mice.

Submandibular glandAlthough morphogenesis of the submandibular gland is knownto start around embryonal day 14, cellular differentiation at thisearly stage of development did not seem to proceed very muchand we could not detect any ISH signals in 14-day and 18-dayembryonal tissues. Even in newborn mice, the gland consistedsimply of ducts and terminal saccules, and no hybridized signal

101Cells expressing cot mRNA

Fig. 3. Expression of cot gene in cultured cell lines. Samples of 1 µgof mRNA purified from colonic adenocarcinoma-derived cell lines(COLO 201, COLO 320DM, LoVo, LS-180, SW 480 and WiDr) andgastric adenocarcinoma-derived cell lines (AZ 521, KATO III andMKN 45) were applied to lanes for northern blot analysis. Afterhybridization with cot probe, the membrane was rehybridized with aG3PDH probe, as a control. All cell lines except AZ 521 expressedcot gene, but its level differed from cell to cell. High expression ofcot gene was detected in SW 480 and WiDr cells.

Fig. 4. Expression of cot gene in tumorous tissues. Samples of 30 µgof cellular RNA isolated from normal parts (N) and tumorous parts(T) of tissues of patients 1-6 were applied to lanes. Expression of cotgene was confirmed in normal tissues of the parotid gland (N1) andcolonic glands (N5, N6). This gene was also expressed in tumorustissues derived from pleomorphic adenomas (T1, T2, T3) andadenocarcinomas (T4, T5, T6), but its level differed greatly indifferent tissues. The cot gene was markedly overexpressed in T4and T6.

was observed. In the tissue of weanling mice, however, cytod-ifferentiation appeared to be completed and serous acini andmucus acini were already formed (Fig. 2B). In addition,striated ducts, which are known to differentiate into granularducts after puberty, were well developed, but they did not showany ISH signals (Fig. 2A).

Sublingual glandDuring early developmental stages, the sublingual gland is notseparated from the submandibular gland. So the absence ofISH signals in the submandibular gland of 14-day and 18-dayembryos and newborns, is also indicative of the absence of cotmRNA expression in the sublingual gland. At the weaningstage, this gland was as well differentiated histologically as inadults, but granular duct cells were not yet found in striatedducts and no positive signal was detected (data not shown).

Parotid glandLike the submandibular and sublingual glands, cytodifferenti-ation of this gland did not progress appreciably in embryos andnewborn mice, and no ISH signal was detected in these stages.At the weaning stage, serous acini were completely formed andthe gland seemed to be as well differentiated as in adults (Fig.2D). As expected, positive ISH signals were detected on serousacini, but not in striated ducts (Fig. 2C).

Gastric glandsThe mucosal layer of gastric glands begins to develop in lateembryonal stages, but no ISH signals were detected in thetissues of 18-day embryos. In newborn mice, small structuresof tubular gland were already formed, but no histologicaldifferentiation of peptic cells or parietal cells was yet observedand no hybridized signal was detected. In the tissue of weaningmice, the mucosal layer was better developed and both typesof cells appeared to be completely differentiated and to be dis-tributed as in adult tissues (Fig. 2F). ISH signals were detectedaround the basal area of the stomach body, as seen in Fig. 2E,and these cells expressing cot were identified as peptic cells,by comparision with an HE-stained serial section (Fig. 2F).

Colonic glandsThe differentiation of goblet cells could be observed in 18-dayembryos. In spite of the early differentiation of these cells, nocot hybridized signals were observed until the weaning stage(Fig. 2G, H). In weanling mice, the mucosal layer of this glandwas better developed and thicker. As shown in Fig. 2I, cotmRNA expression was detected in goblet cells in the basalarea, as in adult tissues.

Other tissues in embryos and newborn micePrevious northern blot analyses suggested that cot gene isexpressed in various tissues of embryos and/or newborn mice,such as the brain, thymus, lung, kidney and placenta. However,in this work no ISH-positive signals were detected in sagittalsections of these tissues.

Expression of cot gene in cultured cell lines For characterization of Cot kinase in vivo, cells that expressCot kinase at a higher level are necessary. We performednorthern blot analyses on cell lines derived from gastric andcolonic adenocarcinomas, since ISH results suggested that cot

gene is expressed in certain cells of gastric and colonic glands.No cell lines derived from salivary glands were established. Asshown in Fig. 3, cot expression was detected in all cell linesexcept AZ 521, although its level varied greatly in differentlines. The SW 480 and WiDr cell lines expressed especiallyhigh levels of this gene.

Expression of cot gene in human tumorsThe ISH results revealed cot gene expression in four types ofglandular cells. Since it was interesting to know whetherdeviant Cot expression was related to tumorigenesis in theseglands, we isolated cellular RNAs from pleomorphic adenomasin the parotid gland and adenocarcinomas in gastric andcolonic glands, and investigated their expressions of cotmRNA. As seen in Fig. 4, low expression of cot gene wasobserved in normal parotid and colonic glands. We also

102 R. Ohara and others

detected expression of this gene in tumorous tissues, but itslevel differed greatly in different tissues. Marked overexpres-sion of cot gene was observed in T4 and T6.

DISCUSSION

In this study, ISH analysis of adult mouse tissues revealed cotgene expression in four types of glandular cells: granular ductcells in the submandibular and sublingual glands, serous cellsin the parotid gland, peptic cells in gastric glands and gobletcells in colonic glands. Then we examined the dependence ofits expression on the developmental stage. ISH analysesshowed that cot gene was expressed only in terminally differ-entiated cells of the above four types. Furthermore, functionalactivation (maturation) of these cells may be necessary for itsexpression, because its expression in goblet cells was notdetected until the weaning stage, although these cells differen-tiated in embryonal stages. As the function of goblet cells is tosecrete mucus to help faecal lubrication, this secretion wouldbe highly activated at the weaning stage. The functional acti-vation of the granular duct cells, serous cells and peptic cellsto produce secreted materials would occur in parallel with theirmorphological differentiation. We could not find any cells inother embryonal and newborn tissues showing ISH signals.

From the results of ISH, we conclude that cot gene is specif-ically expressed in mature glandular cells and that Cot proteinis somehow related to secretory phenomena. It may be involvedin a signal transduction pathway that induces the production ofsecretory materials, or take part in a secretory pathway by whichthe production, transportion and exocytosis of secretedmaterials occurs. Accordingly, we are very interested toperform further hybridization studies on endocrine gland tissuessuch as the pituitary body, pancreas and adrenal gland.

Our previous northern blot analyses indicated that cot geneis expressed in a variety of tissues (Ohara et al., 1993), but inthis study we identified only four types of cells that were cot-positive by ISH. This means that many cells other than theabove four types of cells may express cot gene, but at quanti-tatively lower levels, at which the signal cannot be detected byISH. For example, adult thymus tissue showed as high anexpression of this gene as the submandibular gland (Ohara etal., 1993), but no positive signal was detected on the cells byISH, indicating that all T lymphocytes express cot gene but atlow levels to be detected by ISH. In any case, if Cot kinase isrelated in some way to secretory phenomena, as suggestedabove, expression of cot gene may be widespread because allcells produce secretory materials to some extent.

Various kinds of human cell lines have been tested for cotgene expression, but so far its expression has been demon-strated only in PLC/PRF/5 (hepatoma) and HOS (osteosar-coma) cells (Aoki et al., 1993). In this study, on the basis ofthe results of ISH, we examined cell lines derived from gastricor colonic adenocarcinomas and found that SW 480 cells andWiDr cells express cot gene at about ten times higher levelsthan those in PLC/PRF/5 cells or Hos cells. Thus furtheranalyses may be possible using these cells. We are nowplanning to establish cell lines expressing cot anti-sensemRNA in these cells, for comparision with the parent cells. Inaddition, the finding that most cell lines, except AZ 521 cells,showed expression of this gene suggests that cot gene is orig-

inally expressed in cells of colonic and gastric glands, sup-porting the ISH results.

We also investigated cot gene expression in humantumorous tissues. As seen in Fig. 4, marked overexpression ofcot gene was observed in T4 and T6. This result seems com-patible with findings by transfection assays and tumorigenecityin nude mice that overexpression of this kinase has a trans-forming effect (Aoki et al., 1993). Therefore, in T4 and T6,overexpression of cot gene might contribute to their transfor-mation.

Here, we propose that Cot kinase is related in some way tosecretary phenomena. The cot gene is the first proto-oncogenefound to be expressed specifically in glandular cells, so furtherstudies on the physiological function of Cot kinase should beof great interest.

We thank Dr T. Akiyama for discussion about the manuscript,Messrs K. Morihana and A. Fukuyama for sectioning, Drs Y. Ogawaand M. Aoki for technical advice, the JCRB and Dr Seiki for culturedcells, and the staff of the Department of Oncological Surgery fortissues. This work was supported by grants from the Ministry ofEducation, Science and Culture of Japan.

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(Received 4 July 1994 - Accepted 13 September 1994)