characterization of tissue specific expression of notch-1 in human tissues

Characterization of tissue specific expression of Notch-1 in human tissues

Alfonso Baldi a,1, Maria De Falco b,1, Luca De Luca c, Giuliano Cottone d, Marco G. Paggi d,Brian J. Nickoloff e, Lucio Miele f, Antonio De Luca *,c,d

a Department of Biochemistry and Biophysics “F. Cedrangolo”, Section of Pathologic Anatomy, Second University of Naples, Naples, Italy.b Department of Evolutive and Comparative Biology, University of Naples ″Federico II″, Naples, Italy.

c Department of Medicine and Public Health, Section of Clinical Anatomy, Second University of Naples, Naples, Italy.d Laboratory “C”, Dept. for the Development of Therapeutic Programs, Center for Experimental Research, Regina Elena Cancer Institute, Rome, Italy.

e Department of Pathology and Cancer Center, Loyola University Medical Center, Maywood, IL, U.S.A.f Department of Biopharmaceutical Sciences and Cancer Center, University of Illinois, 833 South Wood Street, room 355,

Chicago, 60612-7231 Illinois, U.S.A.

Received 1 September 2003; accepted 23 January 2004

Available online 09 March 2004

Abstract

Signaling through the Notch cell surface receptors is a highly conserved mechanism of cell fate specification. Notch signaling regulatesproliferation, differentiation and cell death. In vertebrates, putative gene duplication has originated four Notch genes, Notch-1, -2, -3 and -4.They have been implicated in neurogenesis, hematopoiesis, T-cell development, vasculogenesis and brain cortical growth. We haveinvestigated Notch-1 distribution in normal human tissues by immunohistochemistry and immunoblot. We detected widespread expression ofNotch-1 cytoplasmatic staining, with different tissue distributions in the different organs examined. In particular, high expression of Notch-1was detected in the intermediate suprabasal layers, but not in the dead cells at the extreme periphery of stratified epithelia. Moreover, alow/intermediate level of Notch-1 was observed in lymphocytes in several peripheral lymphoid tissues; in particular the germinal centers oflymph nodes showed the most abundant number of positive cells, which appeared to be centroblasts/immunoblasts based on nuclearmorphology. Notch-1 participates in keratinocytes differentiation. We showed by Western blot analysis that Notch-1 level was clearlyincreased in HaCaT cells after Ca++ addition and remained substantially elevated until late differentiation stages. These results suggest thatNotch-1 may function in numerous cell types in processes beyond cell fate determination, such as neuronal plasticity, muscle hypertrophy,liver regeneration, and germinal center lymphopoiesis during the immune response.© 2004 Elsevier SAS. All rights reserved.

Keywords: Notch1; Immunohistochemistry; Human tissues

1. Introduction

The Notch gene family has been the object of extensiveinvestigation in both vertebrates and invertebrates (Green-wald, 1998; Artavanis-Tsakonas et al., 1999; Miele and Os-borne, 1999; Osborne and Miele, 1999). Notch belongs to thefamily of EGF 1-like homeotic genes, which encode trans-membrane proteins with a variable number of cysteine-richEGF-like repeats in the extracellular region. In invertebrates,these motifs participate in cell-to-cell interactions that ulti-

mately regulate differentiation decisions during development(Rebay et al., 1991; Garces et al., 1997). Notch proteins aresynthesized as precursors, which are proteolytically pro-cessed to a heterodimeric, mature form consisting of anextracellular subunit (NEC) that contains multiple EGF-likerepeats and a transmembrane subunit (NTM) that includes theintracellular region, a single pass transmembrane domainand a short extracellular region (Blaumueller et al., 1997).This single transmembrane domain undergoes a proteolyticprocessing event in response to ligand that ultimately liber-ates the Notch intracellular domain (NICD) from themembrane-bound protein (Kidd et al., 1998; Lecourtois andSchweisguth, 1998; Struhl and Greenwald, 1999). NICDthen translocates to the nucleus to affect downstream geneexpression (Levitan et al., 2001). In mammals, four Notchgenes have been described, Notch-1, Notch-2, Notch-3, and

* Corresponding Author: Dr. Antonio De Luca Department of Medicineand Public Health Section of Clinical Anatomy School of Medicine, 2ndUniversity of Naples Via L. Armanni, 5, 80138 Naples, Italy Tel.: +39081 5666008 - Fax: +39 081 458225.

E-mail address: [email protected] (A. De Luca).1 The first two authors contributed equally to this work

Biology of the Cell 96 (2004) 303–311

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© 2004 Elsevier SAS. All rights reserved.doi:10.1016/j.biolcel.2004.01.005

Notch-4 (Robbins et al., 1992; Jhappan et al., 1992; Lardelliet al., 1995). They have been implicated in neurogenesis(Artavanis-Tsakonas et al., 1999; Sestan et al., 1999), he-matopoiesis (Milner and Bigas, 1999), vasculogenesis (Uyt-tendale et al., 2001), and cortical neurite growth (Sestan etal., 1999). When Notch interacts with one of its ligands onthe surface of an adjacent cell, the receptor is proteolyticallyprocessed, and nuclear translocation of the receptor’s intrac-ellular domain results in transcriptional regulation oflineage-specific genes (Egan et al., 1998; Artavanis-Tsakonas et al., 1999; Weinmaster, 2000).

Notch receptors and ligands are widely expressed in mam-mals. Notch-1 and -2 mRNA have been detected in variousorgans of humans and mice (Ellisen et al., 1991; Weinmasteret al., 1992). The same is true of Notch ligands Jagged-1 andDelta-1 in human organs (Felli et al., 1999; Gray et al., 1999;Milner and Bigas, 1999). Notch-3 is expressed in the devel-oping central and peripheral nervous system (Lindsell et al.,1996), thymus (Felli et al., 1999) and pancreas (Apelqvist etal., 1999). Notch-4 is thought to be expressed primarily inendothelial cells during development and adult life (Uytten-dale et al., 1996). Targeted disruption of Notch-1, Notch-2,Jagged-1 or Jagged-2 genes in mice causes severe develop-mental defects or embryonic lethality (Artavanis-Tsakonas etal., 1999; Miele and Osborne, 1999). Expression of constitu-tively active forms of Notch receptors inhibits or delaysterminal differentiation in vitro in many cellular models(Artavanis-Tsakonas et al., 1999; Miele and Osborne, 1999;Milner and Bigas, 1999).

However, it has been demonstrated that Notch-1 is neededfor differentiation in many cell types such as adipocytes(Garces et al., 1997), erythroid cells (Shelly et al., 1999),myeloid cells (Milner and Bigas, 1999; Weijzen et al.,2002a), T-cells (Osborne and Miele, 1999; Robey, 1999; Puiet al., 1999; Yasumoto et al., 2000), murine (Lowell et al.,2000; Rangarajan et al., 2001) and human keratinocytes(Nickoloff et al., 2002) and many others. Overall, the evi-dence indicates that in many contexts Notch activation re-stricts differentiation toward specific cell fates while allow-ing the cell to respond to signals that specify an alternate fate.Several studies have been shown that Notch-1 may be in-volved in the regulation of epidermopoiesis. In murine kera-tinocytes, enforced expression of intracellular (active)Notch-1 induces expression of early differentiation markersand prevents expression of late differentiation markers. Inthese cells, Notch-1 is necessary to induce p21cip1/waf1 ex-pression and thus is able to induce determine the exit fromthe cell cycle which precedes terminal differentiation (Ran-garajan et al., 2001). In human skin, Notch-1 is most abun-dant in basal layers, suggesting that Jagged-1/Notch-1 inter-actions may initiate keratinocyte differentiation (Nickoloff etal., 2002).

Since Notch has multiple conserved domains with thepotential to be protein docking sites, Notch might act as ascaffold to assemble complexes containing components of itsown and signaling pathway and others. As with any complex

signaling systems, physiological functions mediated byNotch are likely to depend on how Notch signals integratewith signals emanating from other pathways (Chu et al.,2002).

In mammals, most of the available data have been ob-tained studying Notch-1 or Notch-2. There is accumulatingevidence that Notch signaling is deregulated in various ma-lignancies (Zagouras et al., 1995; Daniel et al., 1997; Leetha-nakul et al., 2000; Rae et al., 2000; Suzuki et al., 2000;Weijzen et al., 2003). Most of the published work on Notchsignaling in hematopoietic malignancies focuses on lym-phoid leukemias or lymphomas. It is well established thatconstitutive overexpression of active Notch-1, -2 or -3 pre-disposes to T-cell malignancies (Aster and Pear, 2001).Among hematopoietic malignancies, acute myeloid leuke-mia (Tohda and Nara, 2001) as well as Hodgkin and large-cell anaplastic lymphomas (Jundt et al., 2002) have beenshown to overexpress Notch-1, while B-cell CLL has beenshown to overexpress Notch-2 (Hubmann et al., 2002).Moreover, Jagged-1/Notch-1 interactions have been shownto induce proliferation and survival in Hodgkin and large-cellanaplastic lymphomas (Jundt et al., 2002).

These observations suggest that either Notch-1 itself or itsdownstream signaling partners may be potential therapeutictargets in oncological applications. Thus, a thorough under-standing of the tissue and cellular distribution of Notch-1expression in normal human organs is of paramount impor-tance to predict the possible sites of toxicity of putativeNotch inhibitors. In this manuscript we describe a study ofthe expression pattern of Notch-1 in adult human tissuesconducted by immunohistochemistry and immunoblot. Ad-ditionally, to explore the regulation of Notch-1 expressionduring keratinocyte differentiation, we have analysed expres-sion level of this receptor in HaCaT immortalized kerati-nocytes cells by western blotting.

2. Results

2.1. Expression Pattern of Notch in Normal HumanTissues

We determined the approximate level and localization ofNotch-1 in a panel of different human tissues by immunohis-tochemistry (Fig. 1 a). Figure 1 b shows a representativenegative control obtained preabsorbing primary antiserumwith antigen .

We have shown that Notch-1 was widely expressed inmany tissues with mostly cytoplasmatic localization. Differ-ent tissue distributions and/or level of expression were de-tected in the different organs examined (Table 1).

We first investigated the presence of Notch-1 in epitheliaof several organs. In general, epithelial cells, either fromsimple or stratified epithelium, showed positive staining forNotch-1. In particular, stratified epithelia, such as epidermis(Fig. 1 c) and the mucosae of the oral cavity, esophagus,

304 A. Baldi et al. / Biology of the Cell 96 (2004) 303–311

ectocervix and vagina expressed relatively high levels of thisprotein in the intermediate suprabasal layers, but not the deadcells at the extreme periphery. In the skin, Notch-1 distribu-tion was comparable to what previously reported (Nickoloffet al., 2002). As reported by Nickoloff et al. (2002), a highexpression level for Notch-1 was found in hair follicles,sebaceous glands and sweat glands (Tab 1). Interestingly,Zagouras et al. (1995) have shown that only the spinous layerof the cervical epithelium is positive for Notch-1. It is pos-sible that the polyclonal antibody used in our study is moresensitive; or alternatively, that post-mortem phenomenamade Notch-1 more easily detectable.

In the respiratory system, low immunoreactivity was de-tected in the stratified columnar epithelia of trachea, bronchiand adjacent glands. Pneumocytes showed undetectable sig-nal for the protein (Tab 1).

In the gastrointestinal system, intermediate positive cyto-plasmic staining for Notch-1 was found in secretory ductscompared with lower level showed by the acini of salivaryglands. Moderate staining was shown in the epithelia of thestomach and liver (Fig. 1 d) while a low level was seen in thegall bladder and colon epithelia. The endocrine portion of thepancreas showed a moderate level of expression for Notch-1whereas the exocrine portion of the gland was less intense(Tab 1).

In the urinary system, Notch-1 was expressed at an inter-mediate level in all segments of kidney tubules (Fig. 1 e),while a low expression of the protein was seen in the glom-eruli, uroepithelium and prostate (Tab 1).

In the endocrine system, Notch-1 was expressed at anintermediate/high level in the epithelial cells surroundingthyroid follicles (Fig. 1 f). A similar expression level was

Fig. 1. Distribution of Notch-1 in several human tissues.a) Human tissue array (AA4 Normal organs, Superbiochips, Korea), containing 59 normal human adult tissues; b) representative negative control of skinobtained preabsorbing primary antiserum with antigen; c) Notch-1 was expressed in suprabasal layers of human skin; d) moderate level of Notch-1 staining inhepatocytes; e) intermediate level of staining was detectable in kidney tubules; f) high Notch-1 positivity was present in epithelial cells of thyroid follicles; g)intermediate expression level was found in epithelium of proliferative endometrium; h) moderate immunostaining for Notch-1 was present in Purkinje cells ofhuman cerebellum.

305A. Baldi et al. / Biology of the Cell 96 (2004) 303–311

present in the adrenal glands, where staining was more in-tense in the cortical portion than in the chromaffin portion(Tab 1).

In the female reproductive system, we found a low tointermediate expression level of Notch-1 protein in glandularbreast epithelium. Notch-1 was expressed at an intermediatelevel, in particular in the epithelium of both proliferative andsecretory endometrium (Fig. 1 g), in the follicles of the ovaryand in exocervix where an intermediate level of Notch-1 wasfound in the suprabasal layers, in contrast with the basal layerthat was negative for this protein. An intermediate/high levelwas found also in the decidua of placenta, in contrast withlow level showed in syncytiotrophoblast of placental villi(Tab 1). In the male reproductive system intermediate toundetectable cytoplasm staining for Notch-1 was found inthe epithelia: intermediate level of expression was found inepididymis, in contrast with low expression found in thetestis and with undetectable level seen in seminal vesicleepithelium, where only smooth muscle fibers showed inter-mediate positivity for Notch-1 (Tab 1).

Intermediate immunoreactivity for Notch-1 was observedin skeletal muscle cells, as well as in myocardial cells. Unde-tectable signal was found in adipocytes, whereas a low levelof staining was shown in chondrocytes and fibroblasts (Tab1). Moderate level was shown in the arterial endothelium incontrast with an almost undetectable level of expression seenin veins (Tab 1).

In the lymphoid tissue, low/intermediate level of Notch-1was observed in lymphocytes in several lymphoid tissues asthymus, spleen and lymph nodes (Tab 1).

Neurons from different areas of the brain, such as frontalcortex and midbrain, cells of the granular level of the cerebel-lum displayed a low staining for Notch-1, in contrast withPurkinje cells that exhibited a moderate immunoreactivityfor this protein (Fig. 1 h). Perineural and endoneural cells ofperipheral nerves and ganglion cells showed low to undetect-able levels of the protein, as did astrocytes, oligodendroglialand microglial cells of brain tissue (Tab 1).

Table 1Notch-1 protein expression in normal adult human tissues

Tissue Degree ofExpression

SkinHair follicles +++Sweat glands +++Sebaceous glands +++Epidermis +++

Respiratory systemBronchus epithelia +

glands +Pneumocytes +-Mesothelium +

Gastrointestinal systemSalivary glands ++Esophagus epithelia ++

glands +Stomach epithelia ++

glands +Small intestine +Large intestine +Gall bladder epithelium +Liver hepathocytes ++

ductal cells +-Pancreas esocrine +-

endocrine ++Urinary system

Kidney glomeruli +proximal tu-bules

++

distal tubules ++collectingducts

++

uroepithelium +prostate +

Endocrine systemThyroid ++Hypophysis +Adrenal gland cortical ++

chromaffin +Reproductive system

Breast epithelia ++Uterus endometrium ++Salpinx +Vagina epithelia ++Ovary granulosa ++

germ cells ++Epididymis ++Testis Leydig cells +

germ cells +Seminal vesicle +-

Cardiovascular and connective tissueSkeletal muscle ++Smooth muscle ++Myocardium ++Adipocytes ++Fibroblasts +Chondrocytes +Endothelial cells arteries ++

veins +-

Tissue Degree ofExpression

Lymphoid tissueSpleen +Thymus +Lymph nodes +

Nervous systemNeurons +Purkinje cells ++Astrocytes +Oligodendroglia +Microglia +Ependyma +Perineurium +Endoneurium +Plexi choridae +

+−: Low to undetectable expression level (0-10% of positive cells).+: Low expression level (11-20% of positive cells).++: Medium expression level (21-60% of positive cells).+++: High expression level (> 60% of positive cells).


Results obtained with a human tissue array (AA4 Normalorgans, Superbiochips, Korea), containing 59 normal humanadult tissues, were entirely consistent with the data describedon the human tissue obtained from autopsies.

To confirm, by an alternative approach the expressionpattern of Notch-1, we performed a western blot analysis onseveral human fresh tissues. In Figure 2 a representativepanel, including skeletal muscle (lane 1), large intestine (lane2) and skin (lane 3), was shown. These results were consis-tent with the immunohistochemical pattern of expression ofNotch-1 in human tissues.

In order to carefully investigate the expression of Notch-1in lymphoid tissues, serial sections of normal lymphnodeswere immunostained for Notch-1, CD3 (specific for T lym-phocytes), CD20 (specific for B lymphocytes), CD31 (spe-cific for endothelial cells), and CD68 (specific for macroph-

ages). Notch-1 positivity was appeared as intense andwidespread cytoplasmic staining, while nuclei were found tobe negative (Fig. 3 a). The remaining antigens (CD3, CD20,CD31 and CD68) featured their own well-known reactivityprofiles (Fig 3 b-e).

Interestingly, germinal centers showed the most abundantnumber of positive cells, the nuclei of which had the mor-phology of those of centroblasts/immunoblasts and wereCD20+ and CD3-. The centrocytes (CD20 positive) weremostly negative for Notch-1 staining, as were macrophages(CD68 positive), mantle cells and marginal cells (CD3 posi-tive). On the other hand endothelial cells (CD31 positive)were often positive for Notch-1 staining (Fig 3 a).

2.2. Expression of Notch 1 in Differentiating immortalizedKeratinocytes

The evidence described so far indicates the presence ofNotch-1 in many cell types that are not undergoing lineagedecisions, but that do go through an orderly maturation pro-cess. The role of Notch signaling in post-developmentalmaturation phenomena has been studied in detail in humankeratinocytes (Zagouras et al., 1995). In this setting, it ap-pears that activation of Notch signaling can drive the entireprocess of maturation up to and including terminal differen-tiation. Therefore, we investigated whether a well-recognized keratinocyte differentiation signal, namely, in-

Fig. 2. Western blot analysis of Notch-1 expression in several human tissues.A representative panel including skeletal muscle (lane 1), large intestine(lane 2) and skin (lane 3), is shown. HSP70 was used for normalization of theprotein amounts loaded.

Fig. 3. Specific distribution of Notch-1 in human lymphoid tissues.a) Notch-1 positivity was intense and widespread in the cytoplasm of germinal center cells as well as in the endothelial cells; b) CD20 positivity in the cytoplasmof B cells; c) CD3 positivity was present in the cell membrane of normal T cells; d) CD31 specific staining in endothelial cells of lymphoid tissue; e) CD68positivity was present in monocytes and macrophages of human lymphoid tissue. Magnification is 200 X for all the figures.


creased extracellular Ca++ concentration, modulates Notch-1expression in immortalized HaCaT cells. (Fig. 4). Our dataindicate that expression level of mature Notch-1 protein wasmoderate in undifferentiated HaCaT cells (lane 1). Notch-1level was clearly increased in HaCaT cells after Ca++ addi-tion to the culture medium and remained substantially el-evated up to late differentiation stages (lane 2).

3. Discussion

Notch receptors are involved in the regulation of cell-fatedecisions, differentiation and proliferation (Greenwald andRubin 1992; Ahmad et al., 1995; Chitnis et al., 1995; Hen-rique et al., 1995; Myat et al., 1996; Shawber et al., 1996;Greenwald, 1998; Artavanis-Tsakonas et al., 1999;Chaturvedi et al., 1999; Zloblin et al., 2000). In invertebrates,Notch signaling is involved in many developmental cell fatedecisions. This signaling network is conserved in vertebrates,including mammals, where Notch homologues are expressedin dynamic temporal and spatial patterns in many organisms.Multiple functions of Notch-1 signaling are suggested by thewidespread and diverse pattern of expression of its mRNAand protein in vertebrates tissues and cell lines. In particular,several northern blot experiments have been demonstratedthat Notch-1 was involved in several processes such as mes-enchymal cell differentiation (Sakamoto et al., 2002), sper-matogenesis (Hayashi et al., 2001) and osteoblastic cell dif-ferentiation (Tezuka et al., 2002). In addition, Northern blotanalysis performed on rat tissues have suggested that thisgene plays similar developmental roles in mammals (Wein-master et al., 1992). In the present study, we have observedby immunohistochemical analysis that Notch-1 protein has awidespread pattern of expression. In particular, all epithelialcells showed unambiguous immunoreactivity for Notch-1.The expression level in all stratified epithelia was clearlymore intense in the suprabasal layers of the epithelia. Thissuggests a function in guiding the programmed maturation ofepithelial cells, a process that is conceptually separate fromlineage choice. Nickoloff et al. (2002) have recently shown

that activation of Notch signaling by Jagged-1 is necessaryand sufficient to trigger complete maturation and cornifica-tion of human epidermal equivalents. This is consistent witha proposed role of Notch-1 in causing exit from the cell cycleand early differentiation, while simultaneously inhibitinglate differentiation (Rangarajan et al., 2001). Epidermal stemcells express high levels of Delta-1 but not Notch-1 and arethought to retain their undifferentiated state through Delta-Delta homotypic interactions that prevent Notch activation(Lowell et al., 2000). Taken together, these observationssuggest that activation of Notch signaling causes exit fromthe stem cell compartment and eventually early differentia-tion and cell cycle arrest, setting the stage for terminal differ-entiation. Our data suggest that Notch-1 may participate in asimilar process of differentiation in all stratified epithelia ofhumans. Consistent with this hypothesis, we have shown thatNotch-1 expression level increases in differentiated HaCatcells. This specific cell line undergoes modifications thatrecapitulate the keratinocyte differentiation program(Boukamp et al., 1988). The degree of differentiation HaCaTcells reach in vitro is comparable with the suprabasal kerati-nocyte layer in skin sections (Chaturvedi et al., 1999).

Notch signaling regulates all three branches of the cell fatedecision tree: differentiation, cell cycle progression and apo-ptotic cell death (Zloblin et al., 2000). The central nervoussystem is remarkable in that it is an anatomical site whereNotch receptors are clearly expressed in post-mitotic cells,pointing to a biological function of Notch signaling in matureneurons. Expression of Notch-1, -2, and -3 has been observedin mature neurons of various species (Higuchi et al., 1995;Sullivan et al., 1997; Berezovska et al., 1998). We haveshown a low expression level of Notch-1 in different neuronsof human brain. The physiological functions of Notch signal-ing in mature neurons are unknown. It has been suggestedthat Notch activation may be involved in neuronal plasticity(Henrique et al., 1995). Notch expression may protect matureneurons from apoptotic cell death, while defective Notchsignaling due to PS-1 mutations may contribute to neuronaldeath in familial Alzheimer’s disease (Miele and Osborne,1999).

Notch-1 seems to be involved in regulating apoptoticprocesses especially in lymphoid organs. We have shown ahigh expression level of Notch-1 in proliferatingcentroblasts/immunoblasts (CD20+), while centrocytes andmantle cells were rarely positive for Notch-1 supporting aninvolvement of Notch signaling in lymphopoiesis and in thefunction of mature lymphoid cells. In earlier stages of lym-phopoiesis, Notch-1 favours a T-cell fate for pluripotentiallymphoblasts and precludes differentiation towards a B-cellphenotype (Allman et al., 2002). Our data indicate that inperipheral lymphoid organs Notch-1 may participate in laterstages of B-cell differentiation, namely, the clonal expansionoccurring in germinal centers. Recent evidence suggests thatderegulated Notch signaling contributes to the pathogenesisof Hodgkin and non-Hodgkin lymphomas (Jundt et al., 2002)and of chronic lymphoid leukemia (Hubmann et al., 2002).

Fig. 4. Western blot analysis of Notch-1 expression in HaCaT cells beforeand after differentiation.Proteins were extracted from HaCaT cells before (lane 1) and after (lane 2)Ca++ addition to culture medium and analyzed by Western blotting. Undif-ferentiated HaCaT cells show a single band corresponding to the apparentmolecular mass of mature transmembrane subunit Notch-1 (116 kDa). AfterCa++ addition this band was strikingly upregulated. HSP70 was used fornormalization of the protein amounts loaded.


Our data further support the hypothesis that Notch signalingis important in the regulation of lymphopoiesis and, there-fore, in lymphoid cell proliferation.

In conclusion, the pattern of expression of Notch-1 inadult human tissues reveals that this receptor may play mul-tiple roles in several cell types, including cells such as neu-rons, lymphoid cells and skeletal muscle cells, as well assuprabasal cells in stratified epithelia, which do not undergolineage choice. This suggests that Notch signaling may beinvolved in post-choice cell fate determination phenomena,such as neuronal plasticity, muscle hypertrophy, liver regen-eration, lymphopoiesis etc. Further studies will be necessaryto elucidate the details of Notch signaling in diverse normaland transformed cells, including a comprehensive explora-tion of other Notch signaling network components.

4. Materials and methods

4.1. Normal human tissues and cell lines

Normal tissues from autopsy were obtained from the De-partment of Surgical Pathology of the Second University ofNaples, Italy, within the first 36 hours after death. Tissueswere formalin-fixed and paraffin-embedded. For each tissueexamined, at least three specimens from two different indi-viduals were analyzed. A human tissue array (AA4 Normalorgans, Superbiochips, Korea), containing 59 normal humanadult tissues was also used. Human HaCaT immortalizedkeratinocytes were cultured and were induced to differentia-tion by Ca++ addition as previously reported (Boukamp et al.,1988).

4.2. Western blotting

Western blotting on cell lysates or cell fractions was per-formed as described previously (Bagella et al., 1998). Proteinlevels were tested by Bradford assay and normalized byCoomassie blue staining. Moreover the loading and transferof equal amounts of proteins were confirmed after transferredto PVDF membrane by staining the membranes with Pon-ceau Red (Sigma, St. Louis, MO, USA). The goat primaryantibody for Notch-1 (C-20, Santa Cruz, CA, USA) wasincubated with the membrane in 3% milk for 1 h at a1:100dilution and then washed in TBST. The membrane was thenincubated with anti-goat Ig coupled with horseradish peroxi-dase (Amersham, Piscatanay, NJ,USA) and washed inTBS-T. The presence of secondary antibody bound to themembrane was detected using the ECL system (DuPontNEN, Wilmington DE, USA). HSP70 was detected using amouse monoclonal antibody (HSP01, Oncogene Science) for1 h at a1:500 dilution.

4.3. Immunohistochemistry

Immunohistochemistry was carried out essentially as de-scribed previously (De Luca et al.., 2001a; De Luca et al.,

2001b). Tissue sections were quenched sequentially in 0.5%hydrogen peroxide and blocked with diluted 10% normalgoat anti-goat serum (Vector Laboratories, Burlingame, CA,USA). Slides then were incubated at 4°C overnight with thegoat polyclonal immune serum raised against Notch-1 at a1:100 dilution and then with diluted anti-goat biotinylatedantibody (Vector Laboratories, Burlingame, CA, USA) for30 min. Slides containing lymph node tissue were also alter-natively incubated with monoclonal antibodies raised againstCD3, CD20, CD31, CD68 (DAKO, Copenhagen, Denmark)at a 1:50 dilution and then with diluted anti-mouse biotiny-lated antibodies (Vector Laboratories, Burlingame, CA,USA). All the slides then were processed by the ABC method(Vector Laboratories, Burlingame, CA, USA) for 30 min atroom temperature. Novared (Vector Laboratories, Burlin-game, CA, USA) was used as the final chromogen andhematoxylin was used as the nuclear counterstain. Negativecontrols for all antibodies for tissue section were made by: 1)replacing the primary antibody with non-immune IgG; 2)omitting primary antibody; 3) incubating sections with theantiserum saturated with homologous antigen (for this pro-cedure, the antibody was incubated with 10 g/ml of homolo-gous peptide (Santa Cruz, CA, USA) for 48 h). No immu-noreactivity was observed in all the controls.

Two observers (A. Baldi, and A. De Luca) evaluated thestaining pattern of the protein separately and scored eachspecimen for the percentage of positive cells identified. Anaverage of 50 fields was observed for each specimen. Cellswere scored as reported in the legend of Table 1.

Acknowledgements

We thank Mr. Giuseppe Falcone for his contribution to theimages elaboration. This work was supported by SecondUniversity of Naples (A.D.L. andA.B.); NIH PO-1 CA59327(B.J.N.); R01 CA084065 grant and Illinois Department ofPublic (L.M.); I.S.S.C.O. (president H.E. Kaiser).

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characterization of tissue specific expression of notch-1 in human tissues

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