[CANCERRESEARCH55, 1590—1597,April 1. 19951

ABSTRACT

Acute promyelocytic leukemia is associated with a t(15;17) translocalion that generates a fusion product between PML and the retinoic acidreceptor a. Recently, PML was shown to concentrate within subnucleardomains, referred to as nuclear bodies, that are disorganized in acute

promyelocytic leukemia celia. This observation provided the first evidencethat alteration of a nuclear structure may play a role in human pathogenesis. In an attempt to clarify the role of PML and, more generally, ofthe associated nuclear bodies, we used immunohistochemistry to explorethe expression of PML in normal, inflammatory, and neoplastic humantissues. With the exception of endothellal cells and macrophages thatcontain a high amount of PML protein, a weak speckled labeling pafternwas observed in the nucleus of all cell types analyzed. By contrast tonormal tissues, the level of PML expression was considerably enhanced ininflammatory tissues, predominantly around the mononuclear cell infiltrate, as well as during either normal or pathological proliferative states,in particular in tumoral pathology. Surprisingly, in most hepatocellularcarcinoma, a cytoptasmic delocalization ofPML was observed. Finally, thenumber of PML nuclear bodies increased up to twice their normal valueas quiescent cultured cells were StimUlated to grow upon serum addition.Altogether these results strongly suggest that the PML-associated nuclearbodies are implicated both in the inflammatory process and in cell growthcontrol.

INTRODUCTION

APL,3 which accounts for 10% of the acute myeloblastic leukemiain adults, is a clonal expansion of malignant cells blocked at a specific

stage of myeloid differentiation. This type of leukemia is characterized by a specific reciprocal t(15;17) translocation and a particularsensitivity to RA in vitro and in vivo (for reviews see Refs. 1 and 2).Indeed, treatment of APL patients with RA leads to morphologicalcomplete remissions by inducing the differentiation of the malignantclone into mature granulocytes (3, 4). The t(15;17) translocation wasfound to fuse a novel gene, PML, to the RARa gene (5—7),leading tothe production of a PML-RARx hybrid product that retains most of thefunctional domains of parental proteins (8—12).

The mechanisms by which the PML-RARa chimera could exert itsleukemogenic effect as well as the molecular basis for the paradoxicaltherapeutic role of RA in the disease are still poorly understood. It islikely that the hybrid protein could interfere with the functions of theparental RARa and PML proteins. Several lines of evidence suggestthat RARa is involved in the normal process of myeloid differentiation. A retinoic acid-resistant HL6O subclone was shown to contain analtered RARa (13) and introduction of the wild-type receptor restoredthe sensitivity of the mutant cell line to RA (14). A block in differentiation has also been observed in primary mouse bone marrow cells

Received 12/2/94; accepted 2/17/95.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 itt accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by grants from the Association pour la Recherche comic Ic Cancer and theEuropean Economic Community (Biomed and Biotech programs).

2 To whom requests for reprints should be addressed.

3 The abbreviations used are: APt, acute promyelocytic leukemia; RA, retinoic acid;RARa, retinoic acid receptor a; BrdUrd, 5-bromodeoxyuridine;NLS, nuclear localizationsignal.

transduced with a COOH terminus truncated RARa which exertsdominant negative activity (15). Supporting the idea that a modifiedRARa plays a crucial role in APL pathogenesis is the finding that a

variant t(1 1;17) translocation fuses a KrUppel-related gene, PLZF, toRARa (16).

It has been shown that PML-RARa may interfere with othernuclear receptor pathways since it is able to block the differentiationof U937 cells normally induced by the vitamin D3 (17). In this case,a dominant “inactivation―of the heterodimerization RXR partner hasbeen proposed as a possible mechanism of action for PML-RARa(18, 19). When compared to RARa, the PML-RARa hybrid exhibitsaltered transactivating properties but with differences in its phenotypedepending on both promoter and cell types (8, 9, 12). In addition, thefusion of PML to RARa was shown to convert the receptor into anRA-dependent activator of APi transcriptional activity (20), an observation that could be related to the high AP-1 activity usuallyassociated with hematopoietic differentiation.

The function of PML is still unknown. It belongs to a novel familyof proteins characterized by the presence of a C@HC4RING finger,one or two additional Cys/His rich motifs and an a-helical coiled-coilregion (21—22).This latter domain has been shown to preside over theformation of PML homodimers (18 and our own). The family includessome transcription factors such as the RPT-1 repressor (23), the newtA33 protein that is associated with nascent transcripts (24) and the 52kD component of the RO/SSA ribonucleoprotein particle which is anautoantigen in lupus erythematosus (25). More interestingly, threemembers (PML, T18 and RIP) have transforming properties as fusionproducts as a result of chromosomal translocation (26, 27).

There has been growing interest in studying the functional cornpartrnentalization of proteins in the nucleus. Although the role andplace of the different components are relatively well established in thecytoplasm, little is known concerning the function of a number ofsubnuclear structures. Thus far, the best characterized example is thenucleolus, which corresponds to sites of synthesis, processing andassembly of the rRNAs (28). Distinct nuclear domains have also beendescribed as spliceosomes, DNA replication factories and sites ofmRNA transcription (29—32).We and others have shown recently thatPML localizes to discrete subnuclear compartments corresponding tosome type of nuclear bodies (33—35).These macrornolecular structures, which are tightly associated with the nuclear matrix, alsoinclude the SP100 protein, an autoantigen found in 28% of patientswith primary biliary cirrhosis (PBC)(36—37), the NDP55 protein (38)and a 65 kd product (39). In APL cells, the PML-RARa hybrid thataccumulates into aberrant microstructures is able to delocalize some

other nuclear proteins. Interestingly, RA treatment induces a completerelocalization of each of these proteins and, consequently, restores anormal subnuclear organization with apparently intact PML nuclearbodies (33—35).This observation, which offers a striking parallel tothe therapeutic effect of retinoic acid in APL, strongly suggeststhat the disruption of the PML may be directly involved in theleukemogenic process.

Because PML localization has been investigated only in hematopoietic cells, we studied the expression of PML in several normal,inflammatory, and neoplastic human tissues. We show that, while the

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PML Nuclear Bodies Are General Targets for Inflammation and Cell Proliferation1

BenoIt Terris, VéroniqueBaldin, Sylvie Dubois, Claude Degott, Jean-FrançoisFlejou, Dominique Hénin,and Anne Dejean2Department of Pathology. Hdpi:al Beaujon. Clichy (B. T.. S. D.. C. D.. i-F. F.. D. H.), the Unite de Recombinaison ci Erpression Géndtique,NSERM U.163. Institut Pasteur,28 rue du Dr. Rota, 75015 Paris (A. DI. and the Laboratoire de Pharmacologie et de Toxicologic Fondamentales, CNRS UPR 8221. Toulouse (V. B.J. France

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Table1 Im@nunoreactivityfor PML in normal humantissuesStrongModerateWeakVascular

endothelium―keratinocytesHepatocytesMacrophages―

FibroblastsColicglands

Bile duct epitheliumSuperficialkeratinocytes

Lymphocytes―AdipocytesMyocytesGlial

cellsLargeneurons

PML NUCLEARBODY EFFECTON INFLAMMAtiON AND CELL GROWTh

amount of PML was low in normal tissues, a dramatic increase inPML expression was observed during inflammation. Levels of PML

were similarly increased in tumorous states. Moreover, in liver cancers, the overexpression of PML was accompanied by a delocalizationin the cytoplasm. These results show that PML is overexpressed in

distinct pathological situations that are associated with stimulatedtranscription and cell hyperactivity.

MATERIALS AND METHODS

Samples. Immunohistochemistry studies were performed on various nor

mal and pathological human epitheial tissues. All specimens were obtained

from the archival files of the histopathology departments of the BeaujonHospital, Clichy. Resection specimens of 15 patients with colorectal carcinoma, 15 patients with esophageal carcinoma, 23 patients with hepatocellularcarcinoma, 5 patients with cholangiocarcinoma, 4 patients with liver adenoma,5 patients with focal nodular hyperplasia, 4 patients with retroperitoneal

sarcoma, and 5 patients with cervical intraepithelial neoplasia were studied.Furthermore 38 needle liver biopsy specimens obtained for diagnostic purposes were used for this study. Six liver biopsies showed characteristics ofacute hepatitis (3 drug induced, 3 hepatitis B virus positive); 3 of chronicpersistent hepatitis (all 3 hepatitis C virus positive); 17 of chronic activehepatitis without cirrhosis (12 hepatitis C virus positive, 5 hepatitis B viruspositive); 4 of autoimmune hepatitis; 6 of primary biliary cirrhosis; and 3 ofalcoholic hepatitis. None of the patients had received curative treatment. Atleast, to investigate the PML expression in tissue sensitive to hormonalstimulation, 6 endometrial samples were obtained (3 at middle proliferativephase and 3 at late secretory phase). All these specimens were fixed informaldehyde solution for 12—24h and paraffin embedded for histological andimmunohistochemical analysis. In addition, some tumor samples were snapfrozen in liquid nitrogen-cooled isopentane. Five “normal―specimens, freshand fixed, of each tissue were also analyzed. Normal livers were obtained fromuninvolved regions of partial hepatectomy specimens removed for metastaticcarcinoma whereas normal esophagus and colon specimens were providedfrom tissue taken at a distance from the tumor.

Antibodies. The anti-PML polyclonal antiserum had been raised in rabbitsgiven injectionsof a glutathioneS-transferase-PMLfusionproteinas described(33). The 5E10 mouse monoclonal antibody directed against PML and therabbit anti-SP100 polyclonal antiserum were gifts from R. Van Driel (40) andM. Snyder (37), respectively.

Immunohlstochemistry. Sections (5 bun) were cut, floated on poly-Llysine-coated glass microscope slides, and air dried overnight at room temper

ature. Sections from paraffin block were dewaxed in xylene an hydratedthrough graded alcohols to deionized water. To increase immunostaining, thesections were treated with an antigen retrieval solution (10 m@icitric acidmonohydrate, pH 6.0, adjusted with 2 N NaOH) and heated in a microwaveoven at high power three times for 5 mm). Endogenous peroxidase activity wasblocked by a 5-min incubation in 3% hydrogen peroxide, followed by incubation of the specimens in nonimmune goat serum to block nonspecificbinding. After the excess was drained, sections were incubated for 45 mm ina 1:100 dilution of the primary rabbit antiserum (or 1:5 of the hybridomasupernatant) in 3% BSA in Tris-buffered saline. After three rinses in Trisbuffered saline, slides were incubated in 1:100 goat anti-rabbit (or mouse)serum IgG. Subsequently, sections were rinsed three times in PBS, incubatedwith rabbit peroxidase-anti-peroxidase as described by the manufacturer (Dakopatts a/s, Copenhagen, Denmark). Peroxidase staining was performed with3-amino-9-ethylcarbazole as a chromogenic substrate. Then a light nuclearcounter coloration was performed.

Controls consisted of replacement of primary antibody by preimmune serumor use of chromogen alone; these controls were consistently negative orrevealed only cells with endogenous peroxidase activity. To test the specificityof the labeling in some representative sections, the anti-PML immune serumwas applied after incubation with an excess of PML or SP100 proteinsproduced as glutathione S-transferase-fusion products in Escherichia coli.Furthermore, in all experiments, a section of inflammatory liver tissue withhigh PML expression was included as a positive control. The presence ofdistinct nuclear or cytoplasmic staining of normal, inflammatory, and tumoralcells was the criterion for positive staining. The intensity of the staining was

graded as: 0, non staining; +, minimal staining; + +, moderate staining;+ + + , strongly staining.

Cell CUltUreand Synchronization. Humandiploidlungfibroblasts(IMR90) (American Type Culture Collection) were cultured in DMEM in thepresence of 10% heat-inactivated FCS, 2 mMglutamine, penicillin (100 units/ml), and streptomycin (100 @Wml),in a humidified atmosphere containing 5%CO2at 37°C.Exponentiallygrowingcells were arrestedin G0by starvationofFCS during 3 days. They were then stimulated by 20% FCS to allow them tore-enter the cell cycle. Cell cycle progression was followed by measuring

BrdUrd incorporation (41).Immunofluorescence. IMR-90 cells grown on glass coverslips were

washed once with PBS, fixed with 3.7% formaldehyde in PBS for 30 mm at4°C,and permeabilizedfirst with 0,25% TritonX-100 in PBS for 5 mm atroom temperatureand secondly with 100% cold methanol for 10 mm at—20°C.Fixed and permeabilized cells were incubated at 4°Cfor overnight ina humidified atmosphere with the 5E10 antibody (dilution, 1:5). They werethenwashed threetimes with 0.5% BSA in PBS and incubatedfor 30 mm atroom temperature with Texas red-conjugated goat anti-mouse antibodies(dilution, 1:150; Amersham, United Kingdom).

RESULTS

Normal Tissues. The expression of the PML protein in normal

tissues is shown in Table 1. Normal human adult livers displayeda weak positive staining for PML that was confined to a few nucleiof Kuppfer cells and bile duct epithelium on paraffin sections (Fig.lÀ). On frozen sections, due to the increased sensitivity, it was

possible to detect the presence of two or three individual dots in thenuclei of each hepatocyte and endothelial and sinusoidal liningcells (Fig. 1B). On paraffin sections, nuclear staining with PMLantibody was seen on normal human colon, esophageal mucosa,and cervical epithelium (Fig. 3A). In such tissues, both epithelialcells, vascular endothelium, connective tissue stroma, mononuclear inflammatory cells, lymphocytes (almost those situated at theperiphery of lymph nodes), and myocytes showed PML nuclearexpression. No extranuclear staining was observed in any of thesecells. The intensity of the staining varied widely from one cell typeto another ranging from homogeneously dark nuclei in endothelialcells, macrophages, and myocytes to a few dots per nucleus inepithelial cells (Table 1). The latter pattern was observed only onfrozen sections. In all malpighian epithelia, the level of PMLexpression appeared to be more pronounced in regenerative epithelial layers than in superficial layers. Under estrogen stimulation,proliferative endometrium showed a dramatically enhanced cxpression of PML (Fig. 3E) while, in the secretory phase, thenuclear staining of endometrial glands was restricted to a few cells(Fig. 3F).

Inflammatory Liver Diseases. The PML polyclonal antiserum

detected a massive nuclear protein accumulation in portal tracts andlobular parenchyma of inflammatory liver diseases when compared tothe weak immunostaining observed in normal livers (Table 2). In allcases, a strong homogeneous nuclear expression was seen in sinusoidal endothelial cells and Kuppfer cells without any particular correlation with the infiltrating inflammatory cells. Increased diffuse

a Except in normal liver parenchyma where a weak staining is seen.

b PML expression was variable with the highest expression in T lymphocytes.

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Table 2 immunoreactivity of hepatocytesfor PML in normal and inflammatoryliversSpecimen

No. of samples studied Nuclear stainingpositiveNormal

liver tissue 5(Y'Acutehepatitis 66Chronic

viral hepatitis 1717Autoimmunehepatitis 44Florid

biiary cirrhosis 66a

Onlyonfrozensections,a few dotsarevisiblein thenucleus.

PML NUCLEARBODY EFFECTON INFLAMMATIONAND CELL GROWTH

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Fig. 1. Immunohistochemical staining of normal(A, B) and inflammatory (C, D) liver diseases. Onparaffin section, a weak staining is observed in bileduct and Kuppfer cells (A). On frozen section, theanti-PML antibody showed a few discrete nuclearmicropunctations in every cell (B). In contrast,overexpression of PML occurred in chronic viralhepatitis (C) and in primary biliary cirrhosis (D). Astrong homogeneous nuclear staining is seen inhepatocytes and endothelial, biliary and Kuppfercells. The enhanced PMLexpression in hepatocyteswas usually focused in periportal and intralobularareas of inflammation. Arrow, bile duct.

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expression of PML in the nuclei of hepatocytes was found throughoutthe parenchyma of the liver in acute hepatitis and was restricted toperiportal and intralobular areas of inflammation in chronic activeviral hepatitis (Fig. 1C). Portal vascular endothelium, bile duct epithelium, and connective tissue stroma were also intensely positivewhile the level of PML expression was highly variable in inflammatory cells. In all positive cells, the staining was exclusively nuclear

and homogeneous, the nucleoli being prominently visible by exclusion. A strong overexpression of PML in hepatocytes and biliary cellswas similarly observed in non-viral inflammatory processes such asprimary biliary cirrhosis (Fig. 1D) and autoimmune and toxichepatitis (Table 2). In all cases, the high expression of PML wasconfirmed using the 5E10 anti-PML monoclonal antibody (data notshown).

Tumoral Tissues. All cases (15) of esophageal squamous cellcarcinoma and 12 cases (80%) of colorectal adenocarcinomas showedenhanced PML expression compared with normal mucosa. A stronglabeling was similarly observed in the 4 sarcoma cases studied (Table3). The staining was restricted to the nucleus and was homogeneouslybrown with nucleolar exclusion (Fig. 2, A—C).On frozen sections, anuclear punctate pattern was present (data not shown). A strongimmunostaining was also observed in cells of the tumor stroma.

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Table 3 Expression of PML in different humantumorsTumorous

cellsNo.

NuclearandSpecimenTotal positive― NuclearcytoplasmicEsophagus

carcinoma 15 15 150Colorectalcarcinoma 15 12 120Hepatocdllular

carcinoma 23 18 315Osolangiocarcinoma5 4 40Liver

adenoma 4 0 00Focalnodularhyperplasia 5 4 40Cervical

intraepitheial neoplasia 5 5 50Sarcoma4 4 40a

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Whereas esophageal carcinoma showed a diffuse pattern of immunoreactivity involving approximately 80% of the squamous cells, incolorectal adenocarcinoma the PML expression usually varied fromarea to area. In both cases, no correlation was found between the

intensity of the labeling and the state of differentiation of the tumor.Interestingly, the overexpression of PML was not restricted toinvasive components but was also seen in the surrounding dysplasticepithelium of the tumors. In cervical intraepithelial neoplasia, a striking correlation was observed between the level of expression of PMLand the degree of dysplasia that involves the lower third of theepithelium for the grade I to the full thickness for the grade III (Fig.3, A—C).In invasive cervical carcinoma, both neoplastic and stromacells largely overexpressed PML (Fig. 3D). A strong immunostainingwas also noted in cells of the superficial layers infected by the humanpapillomavirus (Fig. 3B).

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Fig. 3. Expression of PML in cervical neoplasiaand in proliferative or secretory endometrium.Comparedwithnormalepitheliumof cervixwhichshowed PML positivity confined to basal regenerative layer (A), in cervical intraepithelial neoplasiaof grade I (B) overexpression of PML is seen inbasal cells occupying the lower third of the epithehum. Cells infected by human papillomavirus insuperficial layers also show positivity. In grade III(C) the full thickness of the epithelium stronglyexpresses PML as well as neoplastic cells and cellsof the stroma in invasive cervix carcinoma (D).During the proliferative phase, endometrial glandsand stroma cellular elements show an intense nuclear staining (E) whereas a few positive cells onlyare noted in the secretory phase (F).

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Of the 23 hepatocellular carcinomas tested, 18 showed an abnormally high expression of PML when compared to the weak stainingobtained in normal adult livers (Fig. 1, A and B). Various patterns ofimmunolabeling were observed in different hepatocellular carcinoma.Surprisingly, in most cases, the pattern was distinctly granular, occasionally ring-like, and diffusely present throughout the hepatocytecytoplasm (Fig. 2, E and F). The intensity of the staining was usuallyincreased along the luminal aspects of pseudoglandular formations.The specificity of the cytoplasmic staining was tested by incubatingsections with preimmune serum or antibody preadsorbed with anexcess of the PML protein instead of the primary antibody. Thesecontrols were consistently negative whereas a persistent cytoplasmicstaining was observed when antibody was preadsorbed with SP100,another nuclear body-associated protein (data not shown). Togetherwith a cytoplasmic positivity, a few dots were present in the nucleus.

The number of them was variable, ranging from 1 to 10 (Fig. 2, E andF). Their large size as well as their doughnut-like morphology wascharacteristic of the nuclear bodies present in PML overexpressingtransfected cells (33—35). In 3 cases, PML expression was strictly

confined to the nucleus (Fig. 2D). The immunostaining for PML wasusually intense in the whole tumor but, in some cases, positivetumoral nodules might coexist with a negative one. The cytoplasmicdelocalization of PML observed in most of the liver tumors analyzedwas detected only when the PML polyclonal antiserum was applied;the anti-PML mAb 5E10 yielded a speckled staining restricted to thenucleus. Moreover, no cytoplasmic delocalization of the SP100 protein was observed in the tumorous hepatocytes as checked by doubleimmunostaining. The specific anti-SP100 polyclonal antiserum revealed only a few intense subnuclear domains and no labeling in thecytoplasm (data not shown).

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Fig. 4. Quantitation of PML nuclear bodies during the cell cycle. A, DNA synthesis in synchronized IMR-90. Cells arrested in G0 by serum deprivation were stimulated by theaddition of 20% FCS. DNA synthesis was monitored by addition of BrdUrd 2 h before the indicated times, at which cells were fixed and stained for BrdUrd detection. The graphindicates the percentage of BrdUrd-positive cells. Each value was determined by counting more than 250 cells and is the mean of two independent experiments. B, localization of PMLby immunofluorescencein synchronized human fibroblasts. G0-arrested and restimulated IMR-9O cells were fixed and stained with the 5E10 mAb (left) and with 4,6-diamidino-2-phenylindole(right). Cells growing exponentially, arrested in Goas well as 8, 16, 24, and 32 h postserum addition are shown. C, histogram showing the number ofPMLdots per nucleusafter serum replenishment. Cells were ranged according to the dots number per cell: less than 10 dots; between 10 and 20 dots; and more than 20 dots at the times indicated after serumaddition. Each value represents a minimum of 200 cells counted and is the mean of two independent experiments.

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Finally, among the five cholangiocarcinoma studied, four displayedstrong nuclear staining of neoplastic biliary epithelium (Table 3). Inaddition, the ductular “proliferation―present in fibrous septa of focalnodular hyperplasia (benign liver tumor) also showed a positivestaining, although the intensity was slightly weaker than in cholangiocarcinoma. In liver adenoma, PML was overexpressed in the nucleiof both vascular endothelial cells and a few compressed hepatocytessituated in the congestive zone.

PML Nuclear Bodies throughout the Cell Cycle. Since PMLexpression appeared to be tightly associated with cell pmliferation, weasked whether the localization ofthe PML protein and its association withthe nuclear bodies could undergo changes during the progression of thecell cycle. After 3 days ofculture in the absence ofserum, more than 98%of the IMR-90 cells were arrested in G0 as assessed by BrdUrd incorporation (Fig. 4A). Following serum stimulation, cells were sampled at 8-hintervals thereafter, and the percentage of cells in S phase was determined. Cells started to synthesize DNA 12 h post-serum addition, and

62% of cells had entered in S phase at 24 h (Fig. 4A). We used indirectimmunofluorescence to analyze the PML labeling pattern in asynchronous cells as well as in G0-arrested or serum-restimulated cells(Fig. 4, B and C). In growing cells, the 5E10 mAb recognized 6—25nuclear bodies per nucleus with an average value of 15; while inquiescent cells, the number was lower with a mean value of 10.Only <2% of cells showed more than 20 dots/nucleus with nearly60% of cells having less than 10. As cells progressed in G1, 8 hafter serum refeeding, a slight increase in cells showing a highnumber of dots was observed; but when cells raised S phase, 24 hafter stimulation, 40% of cells exhibited more than 20 dots/nucleuswhile only 7% had less than 10 bodies/nucleus. Moreover, a weakdiffuse staining pre-existing in G0-arrested cell nuclei appeared tointensify as cells progressed through G1 and S phases (Fig. 4B). Inconclusion, these data show that, athough there is no visiblechange in the PML immunofluorescence pattern in asynchronouscultured cells, a significant increase in both the number of PML

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PML NUCLEARBODY EFFECFON INFLAMMA11ONAND CELL GROWTH

nuclear bodies and the amount of a nonaggregated “soluble―formof PML is consistently observed as cells enter S phase.

DISCUSSION

This investigation documents the occurrence of changes in PMLimmunoreactivity in different pathological processes when comparedto normal tissues. It has been demonstrated that PML is specificallylocalized within nuclear bodies that are also recognized by some

patients' autoimmune sera (33—35).The functional significance ofsuch substructures is presently unknown. A possible association withnucleoli, nucleolar organizer regions and another type of subnuclearstructures, the coiled bodies, is not excluded. A drastic increase in the

number of coiled bodies was evidenced in hormone-sensitive tissues,in viral infected cells, and in stimulated lymphocytes (42, 43). Alter

natively, the PML-associated nuclear bodies may participate in theactivation of transcription of specific regions of the genome. The PMLproteins, known to be expressed in hematopoietic cells (44), is detected at a low level in the nuclei of all cell types analyzed. In basalstates, immunochemistry staining revealed the presence of a fewnuclear dots per nucleus. However, the intensity of expression between the different cell types was highly variable. Endothelial cells,macrophages, fibroblasts, and regenerative cells present in basal layers of epithelium showed an increased PML expression leading to anhomogeneous staining of the nucleus. The low level of PML expression observed in most epithelial cells and, in particular, in hepatocytesprompted us to examine its variation in various inflammatory liverprocesses. Compared to normal livers, acute and chronic viral hepatitis displayed a dramatic increase in PML expression in all cellularcomponents: hepatocytes; vascular endothelium; connective stroma;sinusoidal lining cells; bile duct epithelium; and mononuclear inflammatory cells. While the strong reactivity of endothelial and Kuppfercells appeared to be independent of the infiltrating inflammatory cells,the stained hepatocytes were mainly located to the periportal areas,particularly where inflammation and piecemeal necrosis were more

evident. Hepatocytes in contact to intralobular areas of inflammationwere similarly stained. Such reactivity was also observed in autoimmune diseases (primary biliary cirrhosis and autoimmune hepatitis) asin toxic involvement of the liver secondary to drug or alcohol. Thesedata are consistent with a previous study which demonstrated anenhanced expression of another antigen associated with the PMLnuclear bodies, the NDP55 product, in rat livers intoxicated withcarbon tetrachloride (38). The up-regulation of PML could be secondary to the release of inflammatory mediators such as IFN, tumornecrosis factor a, interleukin 1, and interleukin 6 by infiltratingmononuclear, endothelial, and Kuppfer cells. This idea is supportedby the fact that IFN greatly enhances the synthesis of both the PMLprotein4 and the SP100 autoantigen produced by primary biliarycirrhosis patients (45). Since an overexpression of PML was alsoobserved in some hepatocytes in persistent viral hepatitis, it is likely

that, in this case, a direct viral stimulation or an increased in tumornecrosis factor a expression could be at the origin of the up-regulationseen in the infected cells. This idea corroborates the previous observations that viral infection can induce formation of nuclear bodies(42, 43). Similar observations is made in cervix where human papillomavirus-infected koilocytes showed intense PML nuclear staining.

In both tumoral and nonpathological proliferative states, a highincrease in PML expression was also detected, although to a lesserextent than in inflammatory tissues. The high expression was observed in malignant cells and in regenerative epithelial cells (cryptcells in stomach, basal cells of the cervix or esophageal epithelium).

Moreover, in proliferating epithelium stimulated by estrogens, PMLwas largely overexpressed as observed in endometrial cells. In thisrespect, it is interesting to note that a high increase in the number ofcoiled bodies was shown to correlate with the level of estrogenicstimulation (43). In esophagus and colorectal carcinoma, increasednuclear expression of PML was seen not only in tumoral epithelialcells but also in cells which composed the stroma. Similar observations were made in gastric, thyroid, kidney, pancreatic, and ovariancarcinoma (data not shown). Remarkably, the overexpression of PML

involved not only the invasive component of the tumor but also thesurrounding dysplastic lesions. Furthermore, as in the cervix, theincrease in PML expression levels was correlated to the degree ofseverity of the dysplasia. In all these proliferative states, growth

factors and inflammatory mediators could be involved in the enhancement of expression. However, although the increase in PML expression was observed in all cases of esophageal squamous carcinoma, in3 cases of colorectal carcinoma no staining or exceptional positivecells were detected. Altogether, these results indicate that PML islargely overexpressed in most tumors and in all normal proliferativetissues suggesting that the PML-associated nuclear bodies might befunctionally related to cellular growth.

Interestingly, the PML immunostaining pattern in hepatocellularcarcinoma was clearly distinct from that obtained in other tumors. Anenlargement of the PML nuclear bodies, as found in cells overex

pressing ectopic PML (33—35),together with a cytoplasmic delocalization of the PML labeling into a micropunctated pattern was observed in most (15 of 18) liver tumors. A cytoplasmic PML staining,as revealed by immunocytochemistry, has been previously describedin the case of APL blasts. However, this pattern was shown tocorrespond to an accumulation of the PML-RARa hybrid productresulting from the t(15;17) translocation (44). A similar cytoplasmicdistribution of PML-RARa was observed in stably transfected cells(12, 34) while such a delocalization has never been noted for thenative PML gene product. Although we cannot exclude that thecytoplasmic PML staining observed in tumorous hepatocytes couldresult from a cross-reaction between the anti-PML polyclonal serumand a protein specifically expressed in hepatocellular carcinoma, itseems more likely that this signal may reveal the presence of a PMLisoform lacking the NLS. This hypothesis is supported by the fact thatnumerous PML variant proteins have been shown to derive fromalternatively spliced tanscripts (46). In particular, two types of iso

forms [M56 and M57 described by Fagioli et a!. (46)] that do notcontain the presumed NLS, may well accumulate within the cytoplasm. It is noteworthy that the same isoforms that lack the NLS[encoded by exon 6 (12)J also lack the 5E10 epitope [encoded by exon5 (35)] corroborating our observation that the strong cytoplasmicPML staining detected by the polyclonal antiserum was not visibleusing the 5E10 mAb. Further studies are now required to elucidatethe significance of the differential intracellular localization of thevarious PML isoforms in tumoral diseases. Nonetheless, this workprovides evidence that the implication of PML-associated nuclear bodiesis not restricted to APL but may be extended to other types of humanneoplasia.

ACKNOWLEDGMENTS

We gratefully thank R. Van Driel for the 5E10 monoclonal antibody and M.Snyder for the anti-SP100 rabbit polyclonal antiserum. We also thank J. Seelerfor providing reagents and for reading the manuscript.

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