Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60)

EXPERIMENTAL

CELL

RESEARCH

P9&151-160

(1990)

uclear Sugar-Binding Componen erentiated Human Promyelocytic PATRICEFAGY,ANNIE-PIERRESEVE,MICHELLEHUBERT,MICHELMONSIGNU,AND Centre

de Biophysique Lectines

Molbculaire, Endogekes,

CNRS, Dbpartement 1, Rue Haute, 45071,

INTRODUCTION The existence of sugar-binding proteins (i.e., lectinlike proteins) has been evidenced in the nucleus of reptihan cells [I] and mammalian cells [2-71, and in the macronucleus of a protozoan [B]. Nuclear sugar-binding molecules appeared to be preferentially localized in territories which are enriched in ribonucleoproteins [l4] and are known to be the sites of transcriptional and post-transcriptional events. Likewise, the carbohydrate-binding protein CBP35 was found to be asso-

reprint

requests

should

et

eiated with nuclear bet complexes [7], and the C y to be a heterogeneous ribonucleoprotein ever, sugar-binding components were localized in the rna~~o~~~le~s of Euplotes eurystomus not only in territori ribonucleoproteins but also at the site of tion [B]. Despite these data, the role play lectins remains unknown. Never of these proteins in nuclear corn the sites of fundamental process and DNA replication is intriguing. I tative changes in nuclear lectins acco logical state of cells have been The human tumoral ~romye~o~y~i~ 111 is extensively studied because its rentiation can be induced by treatment with agents [B-17]. Evidence has been a~~~r~~~ated for a crucial role in phosphorylatio~ of n protein kinase C [IB-21], thought to phorbol 1%myristate 13-acetate used to induce the in vitro monocytie differentiation (reviewed in Changes in the oncogene expression are atso [23, 24]. However, the ~~tra~e~~~~ which determine the in vitro cells, with the concomitant ar remain poorly understood. Looking for the function of nuclear lectins and their possible ~~volver~e~t in HLGO cell proliferation and diffe~e~t~at~o~, ~eog~~~o~rot~i~ binding to nuclear components has been investigated during the chemically induced ~ere~t~~~tion of t cells. The present study deals wit the Iocalization an quantitative analysis of nuclear sugar-bin nents, before and during d~~e~e~tia~io monocytes [ 141or into granulocy performed by using isolated a nuclei incubated in the presence of ~~o~~~s~e~n-labelea synthetic glycoproteins, that has proven to be a successful method for analyzing nuclear Hectins ES]. The results reported here suggest that the extranucleolar lectins are involved m the mod~la~,io cell proliferation rather than in the ~b~~oty~~~ tiation of HL60 cells.

The nuclear sugar-binding components (i.e., lectinlike molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 b of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation. 0 1990 Academic Press, Inc.

1 To whom

de Biochimie des Glycoconjugub Orlkans cedex 2, France

be addressed. 151

All

copyright Q 1990 rights of reproduction

0014~4827/90 $3.00 by Academic Press, Inc. in any form reserved.

152

FACY

MATERIALS

AND

METHODS

Cell culture. HL60 cells were grown in suspension in RPM1 1640 medium (GIBCO) supplemented with 10% heat-inactivated calf serum (Sebak), 2 mM L-glutamine, 50 IU/ml penicillin, and 50 wg/ml streptomycin. Cells were incubated at 37°C in a humidified 5% CO, atmosphere and maintained at a density of 2 X lo5 to 1 X 10” cells/ml by resuspending the cells in fresh culture medium every 3 days. Induction of differentiation. Cells used were between passages 16 to 40. Mono&c differentiation was induced by treatment of cells at a density of 3 X lOa cells/ml with 15 rig/ml PMA (phorbol12-myristate 13-acetate, Sigma, St. Louis, MO, U.S.A.). After removing the culture medium, adherent differentiated cells were harvested by shaking in 20 ml cold phosphate-buffered saline (PBS). Granulocytic differentiation was induced by treatment of cells with 1.2% (vol/vol) DMSO (dimethylsulfoxide, Merck). Cells treated with PMA or DMSO were harvested after 12, 24,48,72, and 96 h of treatment. Cell viability was estimated by trypan blue exclusion. Assessment of cell differentiation was performed as follows. (i) The binding rate of the monoclonal antibody OKM-1 (MoAB-OKM,) raised against membrane antigen of monocytes [25,26] was calculated by flow cytometry using indirect immunofluorescence. (ii) Cells were submitted to the nitroblue tetrazolium reduction (NBT Sigma) according to Ref. [27]. (iii) DNA synthesis was judged by [methyl-3H]thymidine (Amersham, sp act 925 GBq/mmol) incorporation in the cells. Briefly, cells were cultured at a density of 3 X lo5 cells/ml with or without differentiation promotors in 96-well microtiter plates (0.1 ml/well) and pulsed with 1 &i/(4 X lo4 Bq) [3H]thymidine per well during the last 6 h of each culture period. Cells were harvested and extensively washed on paper filters by using an automated Skatron harvester (Lierbyens, Norway). Filters were dried and the amount of radioactivity was determined by counting filters in a ASC scintillation liquid (Amersham) with a Beckman LS 7500 scintillation counter. (iv) Morphological changes were monitored in light and electron microscopy. Isolation of membrane-depleted nuclei. Cells were washed twice in 10 mM Tris-HCl (pH 7.5) containing 20 mM KCl, 2 mM CaCl,, 2 mM MgCl,, 0.2 mM spermidine (TKCM buffer) and collected by lowspeed centrifugation (8OOg for 10 min). The pellet was resuspended in 20 ml TKCM buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and Triton X-100 was added to a final concentration of 0.5%. Cells were homogenized and membrane-depleted nuclei were pelleted by centrifugation at 1OOOg for 10 min. The pellet was washed twice in 20 ml isolation medium and collected again by centrifugation. All steps of the isolation procedure were carried out at 4°C within 1 h. Photonic and electron microscopy. Pellets of cells or membranedepleted nuclei were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2), postfixed with 1% osmium tetroxide in the same buffer, dehydrated in acetone, and embedded in Epon 812. For observations in photonic microscopy, 2-pm sections were put on glass slides and stained with toluidine blue. Ultrathin sections contrasted with uranyl acetate and lead citrate were used for electron microscopy. Analysis of sugar-binding components. Fluorescein-labeled synthetic glycoproteins (neoglycoproteins) were prepared as previously described [28]. The following fluoresceinylated neoglycoproteins (sugar-FBSA) were used: cu-D-glucosyl-FBSA (Glc-FBSA), lactosylFBSA(Lac-FBSA), a-L-rhamnosyl-FBSA (Rha-FBSA), a-L-fucosyl FBSA (Fuc-FBSA), a-D-galactosyl-FBSA (Gal-FBSA), ol-D-mannosyl-FBSA (Man-FBSA), and 6-phospho-cu-D-mannosyl-FBSA (ManGP-FBSA). These neoglycoproteins contained 20 * 5 sugar units per molecule, except for Rha-FBSA, which contained 30 f 5 sugar units, as determined by the resorcinol sulfuric micromethod [29]. Observations in fluorescence microscopy were made after incubation of membrane-depleted nuclei in the presence of 125 pg/ml (final concentration) fluoresceinylated neoglycoproteins in the nuclei isolation buffer containing 0.5% bovine serum albumin (BSA) for 1 h at 4°C. Nuclei were washed and collected by centrifugation at 1OOOg for 10 min. Unbound neoglycoproteins were removed with the supernatant.

ET

AL.

Nuclei were resuspended in the isolation buffer and observed with a Zeiss fluorescence microscope fitted with a 490-nm excitation filter and a 520-nm emission filter. For quantitative flow cytometry analysis, membrane-depleted nuclei were incubated in the presence of 12.5 or 25 pg/ml fluorescein-labeled neoglycoproteins. The fluorescence intensity of 10,000 nuclei was recorded using a flow cytometer (FACS 440 Becton-Dickinson, Sunnyvale, CA, U.S.A.). The amount of neoglycoprotein bound per nucleus was calculated according to Ref. [30]. Several controls for labeling specificity were performed as follows: (i) saturation assays by using neoglycoproteins at concentrations ranging from 6 to 125 pg/ml; (ii) homologous competitive inhibition experiments using nonfluorescent neoglycoproteins as competitors at 2 mg/ml (final concentration); (iii) competitive inhibition using free sugars (1 mg/ml) as competitors for labeled neoglycoproteins; (iv) incubation of nuclei in the presence of sugar-free FBSA.

RESULTS

Undifferentiated cells Assessment of differentiation. (Figs. 1 and 2) possessed a large and spherical nucleus containing a slight condensed chromatin network. This network extended throughout the nucleus from a thin layer of condensed chromatin underlaying the nuclear envelope (Fig. 1). A well-developed nucleolus (Figs. 1 and 2) was observed in a number of nuclei whereas others contained several nucleoli (Fig. 2). As seen in electron microscopy, these nucleoli were composed of the three usual elements, i.e., the granular component, the dense fibrillar component, and the fibrillar centers (Fig. 3). However, the latter component was conspicuous and one (Fig. 1) or two (Fig. 3) fibrillar centers were visible according to the section plane. Fibrillar centers were found to be often in contact with the nucleoplasm, as illustrated in Fig. 3. The cytoplasm contained scattered mitochondria and very few Golgi cisternae. Free monoribosomes were abundant (Fig. 1) whereas the rough endoplasmic reticulum was poorly represented. These cells were NBT negative and only 1% were recognized by MoAb-OKM,. Cells treated with PMA began to adhere after 24 h of treatment. Most of them looked morphologically like the initial undifferentiated cells, and only 20% were found to be NBT positive. After 48 h of PMA treatment 90% of cells were adherent to the plastic surfaces and NBT positive. At this stage, morphological changes were observed and cells exhibited the monocytic phenotype. The nuclei were reniform and contained a single nucleolus (Fig. 4) morphologically similar to that observed in undifferentiated cells (Fig. 5). Free monoribosomes were no longer visible and a well-developed Golgi apparatus surrounding the centrosomes was seen in the concave invagination of the nucleus (Fig. 5). Cells treated 72 h with PMA showed a morphology similar to that of cells treated for 48 h, and 90% of them bound MoAb-OKM,. Cell viability was 96% at 24 h, 92% at 48 h, and 80% at 72 h. Treatment for more than 72 h induced degeneration of most cells.

NUCLEAR

LECTIN

CHANGES

DURING

HL6O

CELL

~~FF~~ENTIA~I~~

FIG. 1. Ultrastructure of undifferentiated HL60 cells. A nucleolus (arrow) containing a conspicuous fibrillas center (PC) is visible inside a big nucleus. Note the presence of a network of condensed chromatin (CH) extending throughout the nucleus and Iarge interchromatin spaces (IC). Free monoribosomes (arrow heads) are numerous in the cytoplasm (X10,000). FIG. 2. Semi-thin section through a pellet of undifferentiated cells showing that the spherical nucleus of tbese cells may contain one OI several nucleoli (arrows) (X650). FIG. 3. Ultrastructure of an undifferentiated cell nucleolus. The fibrillar centers (FC), the dense fibrillar component (DF), and the granular component (G) are clearly distinguishable. One fibrillar center is seen in contact with the nucleoplasm (arrovJ). A thin !ayer of condensed chromatin (CM) surrounds the nucleolus (~34,000).

Cells treated for 24 h with DMSO did not present any morphological differences with undifferentiated cells and only 5% were NBT positive. During the following 24 h of treatment, the nucleus of some cells appeared poly-

lobated while other cells still psssessed owever, all nuclei cant cleus. At this stage two hours of

a spherical

nu-

154

FACY

ET AL.

FIG. 4. Semi-thin section through a pellet of monocytes after 72 h of PMA treatment, showing that the cells possess a reniform nucleus containing a single but well-developed nucleolus (arrow) (X650). FIG. 5. Ultrastructure of a cell from the pellet presented in Fig. 4. The nucleolus (NU) appeared to be morphologically similar to the nucleolus of undifferentiated cells. The Golgi apparatus (G) is gathered in the concave invagination of the reniform nucleus (~10,200). FIG. 6. Semi-thin section through a pellet of granulocytes after 72 h of DMSO treatment. Except for one cell (double arrow), the nuclei are polylobated without any visible nucleolus (arrows) (X650). FIG. 7. Ultrastructure of a cell from the pellet presented in Fig. 6. This electron micrograph clearly shows the segmented nucleus (N). Large mitochondria (M), rough endoplasmic reticulum (RE), and cytoplasmic granules (arrows) are visible (X10,000).

locytic differentiation characterized by polylobated and segmented nuclei (Figs. 6 and 7) containing nucleoli so small that they were not seen in light microscopy (Fig. 6). These nucleoli were granule-depleted, as observed in electron microscopy (not shown). Eighty percent of cells were NBT positive after 72 h. Cell viability was 96% until 72 h and 90% after 96 h.

DNA synthesis. DNA synthesis, estimated by [3H]thymidine incorporation (Fig. B), increased in the untreated cell population until 72 h of culture. A slowing down of DNA synthesis occurred between 12 and 24 h of culture in the presence of PMA. After 24 h of treatment, DNA synthesis was found to be half as high as that in untreated cells and to be arrested in cells treated for 48

NUCLEAR

LECTIN

CHANGES

DURING

HL60

CELL

DIFFERENTIATION

possible to know labeled or not.

v

0

24

HOURS

FIG. 8. tion during

FROM

DNA synthesis cell differentiation.

48

72

THE iNITIATION

estimated

96

OF TREATMENT

by [3H]thymidine

incorpora-

h. The rate of [3H]thymidine incorporation was similar to that of untreated cells after 24 h of DMSO treatment. A dramatic decrease in DNA synthesis was observed during the following 24 b of DMSO treatment so that DNA synthesis was very low after 48 h. Ultrastructure of isolated nuclei. Nuclei isolated in the presence of Triton X-100 were found to be devoid of cytoplasmic contaminants and nuclear envelope (Figs. 9 and 10). The material of interchromatin spaces as well as the condensed chromatin remained clearly recognizable (Fig. 9). The nucleolar organization was particularly well preserved (Fig. 10). Localization of nuclear sugar-binding components by fluorescence microscopy. The whole nucleoplasm emitted a bright fluorescence when membrane-depleted nuclei isolated from undifferentiated cells were incubated in the presence of fluorescein-labeled neoglycoproteins. Owing to their eccentric position inside the nucleus, the nucleoli of isolated nuclei are visible in fluorescence microscopy. For instance, they appeared as small spheres emitting a strong fluorescence after staining with acridine orange (Fig. 11). In contrast, when nuclei were incubated in the presence of Rha-FBSA, Glc-FBSA, GalFBSA, Fuc-FBSA, or Lac-FBSA the nucleoli appeared as dark spheres inside the bright nucleoplasm (Fig. 12). Likewise, the nucleolus of monocytes differentiated with PMA was unlabeled (Fig. 13). In this case, however, because the labeling intensity of nucleoplasm was lower than that in undifferentiated cell nuclei, as confirmed by quantitative flow cytometry analysis, the difference between unlabeled nucleoli and labeled nucleoplasm was less striking than that in untreated cell nuclei. The nucleoli of cells going through granulocytic differentiation upon DMSO treatment were not labeled during the first 48 b of treatment. At that point, the polylobated nuclei appeared homogeneously labeled whatever the neoglycoprotein used (Fig. 14). It was not

whether

P55

the very smal

G&anti&hue analysis of nuclear s~g~r-~~~di~g eomponents by flow cytometry. The a ylated neoglycoproteins bound per nucleus were found to be dependent on the nature of sugar residues borne by the neoglycoproteins in nuclei isolated from either untreated dells or DMSOresults of 10 experiments s ity of labeled neoglycopr nuclei was in the followin FBSA or Gal-FBSA > ManFBSA > ManGP-FBSA (Fig. FBSA molecules were abl addition to this result, strongly s~g~est~~gthat the binding of neoglycoproteins to the nuclei is due to t moieties of these proteins, various controls strated the labeling specificity. T s, the amount of neoglycoprotein bound per nucleus 5:found to be concentration dependent, and the saturation of the nuclear

ing of labeled neoglycoprot oteiaas such as GleSA (Figs. l6a an

ments carried out by usi showed that the amount o

free sugars as competitors ound ~eo~ly~ci~roteins was

of Rha-FBSA was 90% (Fig. 17). The ~e~~lyco~rote~~s are acidic moleeul lysine residues of binding of these synthetic proteins per nuclei seem to be related to a cha binding of ~a~6P-F~SA acidic molecule than Glc-F FBSA. Quantitative analysis ~~v~a~~dthat the amount of neoglycoproteins bound per nucleus was low in monoeytes as well as in granulocytes in ~~rn~~~~so~ with the values obtained in E-IL60 cell nuclei ~71 glycoprotein used (Fig. 18 and Table 1’ was especially striking for nuclei of ee PMA for 48 h, which bound fwefoXd 1 tein molecules containing galaetose or (Fig. 18) than did untreated cell nuclei. Decrease of nuclear S SY arrest, and ~~e~oty~~c CO tive analysis was Carrie Rsfrom the same

156

FACY

ET AL.

FIG. 9. Ultrastructure of membrane-depleted nuclei isolated from HL60 cells. The nuclei appeared deprived of cytoplasmic contaminants and nuclear envelope. Nucleoli (NU) are visible. The material of interchromatin spaces is observed in the meshes of condensed chromatin network (arrows) (X8000). FIG. 10. Electron micrograph showing the excellent preservation of nucleolus (NU) in isolated nuclei (compare with the nucleolus of the in. situ nucleus presented in Fig. 1). The absence of nuclear envelope (arrow) is clearly evidenced at this magnification (X20,000).

differentiation. Each kind of nucleus was incubated in the presence of six fluorescein-labeled neoglycoproteins (Glc-FBSA, Man-FBSA, Rha-FBSA, Gal-FBSA, LacFBSA, and Fuc-FBSA). The results of the three experiments were quite similar whatever the neoglycoprotein used. Two examples are presented in Figs. 19a and 19b. During the first 12 h of culture in the presence of PMA, no quantitative changes in the nuclear lectin activity or DNA synthesis were observed. During the same period of culture in the presence of DMSO, DNA synthesis increased while the neoglycoprotein binding remained stable. Between 12 and 24 h of PMA or DMSO treatment, the nuclear lectin activity quickly decreased to

reach a level which remained unchanged during the following 3 days. This early diminution of the lectin activity occurred just before a marked decrease in DNA synthesis in DMSO-treated cells and before the DNA synthesis arrest in PMA-treated cells, which were observed after 48 h. As described above, monocytic differentiation induced with PMA was achieved within 48 h while 72 h was required for granulocytic differentiation. DISCUSSION

The validity of the results presented here is based on three important facts: first, cells were correctly differ-

NUCLEAR

LECTIN

CHANGES

DURING

HL60

CELL

~X~~ER~N~IAT~~N

157

FIG. 11. Membrane-depleted nuclei of HL60 cells observed in fluorescence microscopy after staining with a&dine orange. The nucleoli are visible as small spheres (arrows) emitting a strong fluorescence (X1000). FIG. 12. Membrane-depleted HL60 cell nuclei after incubation in the presence of Glc-FBSA. The nucleoli appear as dark spheres (arrowsj within the fluorescent nucleoplasm (X1500). FIG. 13. Membrane-depleted nuclei of monocyte-like cells after 72 h treatment of HL60 cells with PMA and upon incubation in the presence of Glc-FBSA. Despite the low labeling intensity of the nucleoplasm, dark areas related to the nucleoli are visible (arrows) (X1500). FIG. 14. Membrane-depleted nuclei of granulocyte-like cells after 72 h treatment of HL60 ceils with DMSO and upon incubation in the presence of Glc-FBSA. The nucleoplasms are homogeneously labeled (X1700).

entiated as monitored by several controls; second, nuclear components were very well preserved during nuclei isolation as observed in electron microscopy; third, the specific recognition between the sugars borne by the neoglycoproteins and nuclear lectins has been clearly established. As far as this specificity is concerned, it is noteworthy that a marked diminution in neoglycoprotein binding occurs when free sugars are used as competitors. Keeping in mind that free sugars have much less affinity for lectins than glycoproteins (see, e.g., Ref. [31]), the decrease in neoglycoprotein binding obtained under such competitive conditions must be considered highly significant. The fact that Rha-FBSA is the best ligand when mammalian cell nuclei are used, although a-l-rhamnose is not known to be a component of any mammalian glycoconjugate, has already been discussed in detail [3, 311. On account of these preliminary remarks, the biological meaning of results obtained in this work can be examined. Surprisingly, the nucleoli of undifferentiated HL60 cells as well as the nucleolus of in vitro differentiated monocytes do not exhibit any detectable sugar-binding

molecules, unlike the nucleoli of ceils st 4, 81. The excellent preservation of nuclei excludes the possibility of an arkifact due to an impairment of nueleolar structures during nuclei isolation. Work is currently in progress to elucidate the meaning of this nucleolus behavior. owever, whatever its significance, this absence of n eolar labeling was found to be operationally very us analyze the sugar-binding activity as the extranucleolar territories. These territories are known to include the transcrip ly inactive condensed chromatin and interchro spaces containing transcribing genes and ribo~~~~e~~~ote~~ complexes packaging RNA molecules. to the large volume occupied by interchromatin condensed chromatin areas in the cells studi fact that the whole extranucle peared labeled allows us to assum tin activity detected is as of interchromatin spaces. component of i~terchromat~~ spaces and because it is known to recognize terminal ~-~~~a~a~t~~~de Tesidues

158

83E z -

FACY

0 0

10

20

30

40

L

NEOGLYCOPROTEIN

(pg/ml)

FIG. 15. Representative experiment showing that the amounts of fluoresceinylated neoglycoproteins bound per nucleus (fg/nucleus) is dependent on the nature of sugar residues and on the concentration of the neoglycoproteins used. Background (binding of FBSA) was substracted to give the values shown. Background was 10 fg per nucleus.

[7, 91, CBP35 is likely included among the lectins detected by using Lac-FBSA bearing a P-D-galactoside residue in a nonreducing terminal position. However, leaving aside Rha-FBSA, Gal-FBSA, Glc-FBSA, and ManFBSA which contain D-sugars in an a-anomeric configuration are far better ligands than Lac-FBSA. Therefore, most of the lectins detected in the nucleoplasm of HL60 cells are not related to CBP35. As shown by quantitative analysis, the neoglycoproteins binding is higher in proliferative HL60 cells than in cells differentiated either into monocytes or granulocytes. The lowest level of extranucleolar lectin activity was reached as early as 24 h in cells treated with DMSO or PMA. A selective loss of soluble lectins during isolation of nuclei from differentiated cells alone is unlikely, since the nuclei of undifferentiated and differentiated cells were isolated according to the same procedure. Therefore, this decrease in the sugar-binding ability of nuclei can be reasonably thought to be related to functional changes in cells, as suggested by the results of the present study. Indeed, the decrease in lectin activity occurred just before the DNA synthesis slowed down, while 2 days separated the lectin decrease and the granulocytic differentiation. This temporal correlation suggests that extranucleolar lectins might be involved in the modulation of DNA duplication rather than in the phenotypic differentiation program per se, in particular when differentiation is induced by DMSO. This concept is in agreement with data indicating that the control of growth arrest and the phenotypic differentiation program could be uncoupled during granulocytic differentiation induced by DMSO [32]. Other data showing quantitative changes in nuclear lectins according to the mitotic activity of mammalian cells [3, 61 also support an involvement of these proteins in DNA duplication. The colo-

ET

AL.

calization of lectins and sites of DNA duplication in a lower eucaryote [S] may also be relevant to such a function. However, taking into account their preferential association with ribonucleoprotein complexes, nuclear lectins have been thought to be involved mainly in posttranscriptional events and RNA transport [3, 331. The two hypotheses may therefore appear to be conflicting. Nevertheless, a possibility which would reconcile the two functions may be that lectins, associated with hnRNPs or being hnRNPs themselves, mediate posttranscriptional control of the processing and/or transport of mRNAs specific for synthesis of proteins required for cell proliferation. Recent data indicating that a subset of hnRNPs could modulate in this manner the cell proliferation during differentiation of 3T3 cells and normal keratinocytes [34] support this concept. Moreover, the lectin CBP35 seems to be cotranslated with mRNA in the form of ribonucleoprotein complexes [33]. Among several questions still unanswered is how nuclear lectins act in such a process. Indeed, the decrease

0

z z1500

A-A a--a

?

L

t; z

-;;

Glc-FBSA Glc-BSA+Glc-FBSA

10

40

50

NEOGLY?OPROTElN3~pg/ml) 2

-glOOO-

3 c

b a

II

l -*Man-FBSA 0-OMan-BSA+Mon-FBSA

m 750.

NEOGLYCOPROTEIN

&g/ml)

FIG. 16. Mean values i SE of three independent competitive experiments. Nuclei were incubated in the presence of different concentrations of fluorescein-labeled neoglycoproteins with or without an excess of the same unlabeled neoglycoprotein (2 mg/ml). (a) Inhibition of Glc-FBSA binding. (b) Inhibition of Man-FBSA binding.

NUCLEAR

LECTIN

CHANGES

DURING

HL6O

CELL

DIFFERENTIATION

159

1 n [II I

Non competitive Competitions competitions

with with

Lectin Activity of Nuclei from ~~d~~ere~t~~t~~ and Differentiated HL6Q Cells Evaluate by the Amounts of Fluorescein-Labeled Neoglyccprotein ound per Nucleus

conditions neoglycoproteins free sugars

Neoglycoprotein Neoglycoproteins

HLGO

a-L-Rba-FBSA a-~-Glc-FBSA

1973 k 275 915 k 108 878 +- 115 252 i 51 229 f 58 51+ 15

WD-&&FB%

a-~-Fuc-FBSA Lac-FBSA a-D-ManGP-FBSA

FIG. 17. neoglycoprotein as competitors

Representative (2 mg/mi; for labeled

0

UNDIFFERENTIATED

HL60

CELLS

(a)

2e I

2 2 EY I FI

,"

6 P

2 L

I

:: _I

FIG. 18. Comparative analysis of the nuclear lectin activity in undifferentiated HL60 cells (a), 48 h PMA (b); and DMSO (c) treated cells by using labeled neoglycoproteins at 25 @g/ml. (a, b, c) Mean values of 10 experiments (see Table 1).

PMA”

808 f 237 i 198 + 70 k 59 I NDd

of molecular interaction between nuclear glycoproteins as recently quently, lectins could be prese tiated and differentiated cell nu tins would be no longer recognized

DMSO’

113 71 52 19 62

1203 f 207 512 i- 95 561 + 102 156 k 21 137 i 23 ND

nuclear

lectins

and

wever, these lecby exogeneous neo-

i; al 20 z a 6 g 15 8 3 : Y

3 P

Cells

per nucleus”

a Mean values of 10 experiments. * Upon 48 b treatment. ’ Upon 72 h treatment. d Not done.

experiment using either unlabeled 25 )&f) or free sugars (1 mg/ml; 5.5 mM) neoglycoproteins (25 pglml).

in the sugar-binding ability of undifferentiated cell nuclei may be due to several reasons: (i) Nuclear lectins might be present but inactivated with regard to sugar binding by a post-translational mechanism. (ii) According to the hypothesis that some lectins might be involved in RNA transport, they shuttle between the nucleus and the cytoplasm. Most of them would be localized in the cytoplasm in nondividing cells, while they would be more abundant in the nucleus when the cells are proliferating, as suggested by the behavior of the lectin CBP35 in quiescent and reactivated 3T3 fibroecause the existence of nuclear glycoproteins is now well established (reviewed in Ref. [35]), some nuclear functions might be modulated by a system

boundlfg

10

0

12

HOURS

24

36

48

FROM THE INITIATION

60

72

84

OF TREAi-MEhT

FIG. 19. Comparative analysis of lectin activity decrease, DNA synthesis, and cell differentiation. (a) PMA-treated cells (the phenotypic differentiation occurs after 48 h). (b) D&&SO-treated cells (the phenotypic differentiation occurs after 72 h),

160

FACY

ET

glycoproteins because they are already bound to endogeneous glycoproteins in differentiated cells. In fact, owing to the potential ability of nuclear lectins to reversibly interact with nuclear glycoproteins as different as DNA polymerase 01,transcriptional factors, and nuclear pore proteins (reviewed in Ref. [36]), nuclear lectins could be multifunctional regulators. Recent isolation of lectins from HL60 cell nuclei (unpublished data) will allow us to obtain monoclonal antibodies raised against these proteins. The use of both antibodies against epitopes different from the sugar-binding sites and labeled neoglycoproteins will constitute a suitable procedure for providing further information on the functional role of nuclear lectins. The authors are indebted to Dr. A. C. Roche for providing HL60 cells kindly given by Dr. Le Flock (RhGne Poulenc). We thank Mrs. M. L. Welter for her valuable help in the management of the flow cytofluorometer and P. Bouchard for the synthesis of neoglycoproteins. We thank also H. Labbe for his skillful technical assistance, G. Coste for his excellent photographic work, and Mrs. G. Dolmeta for carefully typing the manuscript. This work was supported by CNRS (LP 4301) and by a grant from ARC (6465) and FNLC. Annie-Pierre S&e is Chargee de Recherche INSERM, Michel Monsigny is professor at the Universite of Orleans, and Jean Hubert is Directeur de Recherche CNRS. Patrice Facy received a fellowship from Minis&e de la Recherche et de la Technologie.

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AL. 11.

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12.

Collins, S. J., Ruscetti, F. W., Gallacher, R. E., and Gallo, R. C. (1978) Proc. Natl. Acad. Sci. USA 75, 2458-2462. Huberman, E., and Callahan, M. F. (1979) Proc. Natl. Acad. Sci. USA 76,1293-1297. Rovera, G., O’Brien, T. G., and Diamond, L. (1979) Science 204,

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868-870. 15.

Breitman, T. R., Selonick, Natl. Acad. Sci. USA 77,

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Hoessly, M. C., Rossi, Res. 49,3594-3597.

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Sokoloski, J. A., Beardsley, Cancer Res. 49,4824-4828.

18.

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