~pment, 38 (1987) 207-217 aad Ltd.
ACCHARIDE MODULATION DU LECTIN BINDING
207
~ERENTIA-
PAUL L. MANN*, ISABEL LOPEZ-COLBERG and ROBERT RO O. Department of A natomy, University of New Mexico, School off iMedicine, A fU.S.A.)
:M87131
(Received August 5th, 1986) (Revision received December 9th 1986) SUMMARY tges in their uantitative and qua IMR-90 human lung fibroblasts exhibit quantitativ~ ltact inhibicell surface oligosaccharide expression as a result of low-density lo level (PDL). [ mcanavalintion of growth and changes in population doublingg level ~ctnus shown A (CON-A), wheat germ agglutinin (WGA)and Ricinus !cinus communts communi (KUA-120)Pwas sh gnificantly to decrease by 20--40% as the cells advanced througlh their lifespan (but signific~ with senescence, which ~hich ooccur before morphological manifestations commonly associated associa at approximately PDL 45). The binding of Dolichos b~iflorus agglutinin (DBA)incre increased ntitation of by 100% over the same time interval, These changes were detected by quantitatio iotin-avidinFITC conjugated lectins on single cells and the use of~a sensitive analytical biotin-av These studies were perfor mrformed enzyme amplification assay on whole populations of cells. c¢ role for on randomly growing cultures. These results suggest an aT important and dynamic roh cellular senescence. the cell surface oligosaccharide in growth control and ce
K e y words: Cellular senescence .~scence, Oligosaccharides; Lectins; Differentiation
INTRODUCTION The cell membrane and md its external surface functions as an organelle which med mediates le interior of the cell as well as serving as a major compone mnent of signal transduction to the the complex regulatoryt mechanisms which controls growth and differentiation 111. The model system for the study of these control mechanisms should consist of both
*Address all correspondence'e and reprint requests to: Dr. P.L. Mann, BRF 120, Dept. of Anatomy, Anal School of Medicine, Univ. off New Mexico, Albuquerque, NM 87131, U.S.A.
0047-6374/87/$03.50 Printed and published in Ireland
© 1987 ElsevierScientific Publishers Ireland Ltd.
cell-contact such as low.density growth inhi d a terminal growth point. The f lditions are each division cycle; the last occurs requency or cell's history. We have chosen the ' Lel for these gested as a criteria. Cellular differentiation h growth control and cellular senescen, structures have been studied in ce dtion [3,4], increasing irnport~ and differentiation [ 5 - 7 ] . They are assuming increasir ~r-associated cell surface changes [8-10]. At the molecular level studies stt have lat the same gene produces potentiating and suppressing signals. The Th functioJ e lies in the glycosylation of the peptide [11,12]. Fibroblasts have been use strate lectin binding changes with advancing PDL [13-16]; however, howe these d very high concentrations of lectin, which tends to form mlcro-crystallit, micro o lacked an analytical-data capability which is required for the study stu~ of the' ,~s of growth control, i.e. low-density and contact-inhibition of grc rowth as ellular senescence model. This study employs two types of assay. The first is a FITC c ctin uptake analysis of the IMR-90 surface at various PDLs and under un variot aditions. By quantitating the fluorescence on a single cell basis it is i: possible 'or vari~ variation ~ossible to control tot ~re. The second assay is popula caused by sub-populations of ceils within the culture. Lationamplification syste dependent but uses the increased sensitivity of the biotin-avidin bio stem to gain insights into low-density growth control. In both assays the following lectins w e r e (P used: wheat germ agglutinin (WGA); Dolichos biflorus (DBA); peanut agglutinin (PNA); ON-A), and Ulex europaeus (UEA I); Ricinus communis (RCA-120); (RCA-121 Concanavalin A (CON-A) soybean agglutinin (SBA). The nominal carbohydrate specificities for these lectins are: lactosamine WGA, N-acetyl-D-glucosamine (GlcNAc) and sialic acid; DBA, b-N-acetyl-D-galactosa alactosamine (Gal BI-3 GalNAc); UEA (bGalNAc); PNA, D-galactosyl-b-(1-3)-N-acetyl-D-galact ~A-120, b-D-galactose (b-D-Gal); CON.A, a-o-glucosyl, marmosyl mare I, L-fucose (L-Fuc); RCA-120 ~hotometry -N-acetyl-D-galactosarnine (a,b-GalNAc). Quantitative photon (a-D-Glc,Man); SBA, a,b-N-ace1 of FITC-lectins showed binding decreases of WGA, CON-A, and RCA-120 as the cell respan. A large increase in DBA binding occurred over the same advanced through its lifes :e lectin-dependent in terms of the kinetics of onset and pre 9recede time. These changes were entation of senescence. Similar dynamic surface changes w e r e the morphological presentation seen during contact-inhitbition and low-density induced growth control regardless of PDL. M A T E R I A L S AND METHODS
Cell culture ang fibroblasts, IMR-90s [17], were obtained from the Institute Insl Human embryonic lung were Camden, NJ, at PDL 12.13 and PDL 40.87. All cultures for Medical Research, maintained in McCoy's 5A medium (Flow Labs) supplemented with 10 mM Hepes I~puffer rn Calf Serum, and penicillin/streptomycin (Flow Labs) at 100 1( IU at pH 7.0, 20% New Born
209 Cultures were passaged in 75-cm 2 ti: a air atmosphere at 37°C. Once sta~ wn to approximately 60-80% confl~ ty of growth pattern and reliabh ures were subcultivated at 1 × 106/f cultivated at 2 X 106/flask. Cultures "ceding at 2-day intervals. To passage each culture, cells were rinsed with Ca//~ /Mg free pt (PBS, pH 7.0), followed by a 2 - 5 rain exposure to 5 ml n of 0.25 all cells had detached, 5 ml of complete medium was added ad~ to the fuged at approximately 200 g for 5 min, washed 1-3 times tiJ with '~ a hemacytometer. Staining aliquots with Trypan Blue p9rovided al Cell stocks were assayed for mycoplasma using the fluorome during the experiments, and later by the Mycotrim-T 'cotrim-TC assay s! (Dupont, Inc.).
flasks (Lux) haetics were subcultivaion of cell cultures at ed approxi'fered saline 37°C. After were centricounted on of viability. Je regularly aa Biologics
Fluorometric analysis of single cells .gl samples IMR-90 cells at varying PDLs were seeded in 20-tt ~) onto previously autoclaved Carlson 10 spot slides (Carlson Inc.~). Slides w t humidified Petal dishes and incubated at 370C for 2 - 1 6 h. After Afte~ visual inspection, cultures were washed 3 times (5 min each) with PBS, fixed in 2.5% formaldehyde (Tousimis) in PBS ~erature, and washed with1 PBS. After containing 5% b-D-glucose for 20 min at room temper~ st~ 2 - 1 6 h of incubation in 0.1 M glycine (or 0.1% BSA in i PBS), the cultures were stained with the following FITC conjugated lectins (Vector Labs): I CON-A, SBA, WGA, RCAF 120, PNA, UEA-I, and DBA. Optimal concentrations of c each lectin were determine termined by titration. Generally 5/ag/ml gave an optimal signal]no~ tnoise ratio. All lectins were diluted dilut in a 0.1% solution of BSA in PBS. Separate control cultures cult were incubated in BSA//PBS. humidifier boxes, washed and mounted mount in Cultures were incubated for 90 min at 37°C in humidif Aquamount (Shandon) covered with a coverslip and allowed to dry. All opera ~erations involving the FITC-lectins :ins were done in subdued light in order to minimize pt~hotobleaching. Photometric analyses of single cells were performed on a Leitz microscope equi qmpped with a UV epi.illuminator tor and a MPV fluorescence photometer. The photometer w a s zeroed mechanically and the 100 mV reading was adjusted while viewing an early pa ~assage cell stained with CON-A. A. A collimating iris in the photometer head was adjuste ted to provide single cell readinags. Because of the large variation in cell size, especially between bet, early and late passage cultures altures, either: (a) two settings of the iris diaphram were used us in which case individual correction ions for background fluorescence were made; or (b) on ~rrections one iris setting to accomodate the he increased size of the late passage cells was used for all readings. read Subsequent readings were ~re transformed into relative fluorescence (R f) by the following follo formula: Relative fluorescence (Rjf ) = (.4 -- ~ ) / ( C -- B ) X 100
ectin bound to the cell, B is the resp~ bitrary maximum
lstained cell
nents included the use of unlabelle( use of both specific and non-specif the lectin binding.
',ompete for harides and
Quantitative analysis of whole populations of cells into sterile (10{ Fibroblast cultures (IMR-90) at various PDLs were seeded s~ te medium. 96-well culture trays at concentrations between 10 a and 106/n After 16 h of culture at 37°C, cells were harvested, medium and three as des 9usly. After washes in PBS were followed by a 20-rain fixation step st at 37°C in further washes and a glycine-block step, trays were incubated i bs). Followlated lectin,, varying concentrations (0.625-10 mg/ml) of biotinylal buffer (0.9%1 Tris, 0.05% hag further washes in enzyme immuno wash (EIA)buff at 37°C with a Tween 20 (pH 7.4)) ceils were cultured for 90 min 3~ Lat 5/xg/ml. ~aline phosof bic After three EIA washes cells were incubated with 100/al 100 e incubated phatase, 5 U/well for 90 rain at 37°C. Following a final'three wasl lviannnelm) im suostrate ~ulIer t ~uffer (10% in 200 p.l of p-nitro-phenylphosphate (Boehringer Mannheim) Mm 0-120 diethanolamine in water, 0.01% MgC12, 0.014% ZnC12 pH 10.2) at 1 mg/ml for 330Multiscan rain at 37°C. The optical density at 405 nm (OD/405) (OD/40 was determined in a Multi reader (Flow). Results were corrected for backgroun round and/or non-specific uptak )take by treating controls with all reagents except the biotinylate lated lectins. RESULTS AU cultures were maintained, as nearly as possible,,under optimal growth condittions. ',ration times (Tg) for IMR-90 cells as a function of PDL. The TI1 Tg Figure 1 shows the generation mriod of shows a relatively stable value from PDL 20 to PDL 32, followed by a perio 5. After another relatively constant growth period to PDL 45 4~ the increasing Tgs to PDL 35. ceils rapidly senesce. 9ecific he staining patterns of the four lectins which exhibited sp~ Figure 2 illustrates the and (d) binding to the IMR-90s:: (a) FITC-CON-A; (b) FITC-WGA; (c) FITC-RCA-120; an~ FITC-DBA. These were the only lectins that appeared to specifically stain the cells, ins tested represent all the major specificities. The staining itself although the seven lectins nificant rose or non-existent, i.e. there does not appear to be a signif appears to be either intense similar ecific labelling. Because the uptake of staining was very si~ "noise" level of non-spec staining on Ihe photographs in Fig. 2 were also similar. The DBA stainir the exposure times for the culture early PDL cells was onl~y slightly above background and therefore a late passage cu was used. The staining was generally of the form observed for other cell surface rmolemay be cules, but not as diffuse as some documented integral membrane proteins. This ms due to the more amorpt,hous nature of the oligosaccharide staining entity. In some cells
211 nerotion Time with PDL in I M R
A
14C
v
E I-c
120 I00
0 (D e-
t9
8O •
60
00
•
4O 20 20
1' 23
,
,
J
I
26
29
52
35
!
38
!
!
!
!
41
44
47
50
Passoge Doubling Level (PDL.) desc Fig, 1. Correlation of generation time (Tg) with PDL in IMR-90 IMR-9( cells. Ceils were prepared as described in the Materials and Methods section. Calculation of Tg was as follows: fi log (final/initial conc.) A= log 2 culture time h Tg= A
a typical "ring" patter ,attern of which were not as flattened as most of the fibroblasts, fibrobh staining was observed indicating a surface labelling. Controls with unlabelled hlectin te competitors indicated that the staining was specific. Genex Generally, and specific carbohydrate 10 mM solutions of specific ,~cific monosaccharides inhibited the specific lectin binding, c o r n pletely and concentrations ons of unrelated monosaccharides as high as 20 mM did n o t show any inhibition. Mild r [d ttrypsin treatment did remove the FITC staining, also indicating indic~ an external cell surface reaction.
Single cell analyses In order to determine ine quantitative differences in lectin binding associated with terminal differentiation , photometric readings were performed on individual cell cells. It should be stressed thatt in these experiments whole individual cells were comF~ared independent o f their size. ~timum Lze. In a series of experiments designed to determine opth lectin concentration for staining the photometer was adjusted to 100 mV (i.e. 87 87% of full scale deflection) at the highest concentration used, i.e. 20/ag/ml. The lectin cot concentration which gave values es just below saturation was considered optimal, about 5 #t uglml. Lectins which did not stain specifically, SBA, UEA I, and PNA, showed no uptat ,take at
~
g
N
I
c o n c e t r a i o n s < 2 0 ~tg/ml.
I//
l~erimeenvtiaklles
Fig. 2. Fluorescence staining pattern of FITC-lectins on IMR-90 ceils. Ceils were cultured on Ca Carlson 10 spot slides and preparedt as described in the Materials and Methods. Cells were stained with: witt (a) FITC CON-A; (b) FITC WGA A; (c) FITC RCA-120; and (d) FITC DBA, at 5 ug/ml.
nl. Above 2 0 / l g / m l some of these lectins showed very high h of uptake which was not concentration dependent and probably resulted from nonspecific crystallite preci]~ pitation on the cell surface. Because of the inter-experim~ variation 2 or 3 lectin concen 3ncentrations were used with each experiment. Experiments these show no significant it PDL-dependent relationship in the ability to saturate the cell surface.
213
JORESCENCE (R/) OF SINGLE CELL~ EL (PDL)
AND LATE
in the Materials and Methods section fro was calculated as a percentage of the o meter was adjusted to an arbitrary 100 m
readings from s. The lectins ceils stained
Lectin
P D L 29 IMR-90s
rMR-90s
CON-A WGA DBA RCA-120
100 116 26 136
7 5 I 5
_+9.2 +3.2 + 3.0 +4.7
Table I shows data from experiments c o m p a r i n gthe lectin PDL 29 and PDL 41. Lectin concentrations of 5 #g[rml were from single cells (approx. 30/group) were compar( )ared. Redu observed for CON-A ( - 1 7 % ) , WGA ( - 8 0 % ) , and RCA-120 1 increase in DBA (+300%) binding when PDL 41 cells were cot
IMR-90s at
te responses nding were well as an ) L 29 IMR-
90s. Using a Student's t-test and an analysis of variance va " the differences; for tor WGA, V th~ RCA-120, and DBA were all significant at the 1% level. Table II shows the kinetic aspect of these binding alterations. al R f values were obtained obt~ from cultures of PDL 2 5 - P D L 44 cells. There was no significant change in the R / •vvalues from PDL 25 to 35. Therefore, in order to have a stable stabl base for data subsequent Rfs were calculated as a percentage of the PDL F 25 Rf. The appear to be lectin-dependent. CON-A shows a Pr0grressive decrease RCA-120 reaches a minimal R / a t PDL 39 and there thereafter increases
comparison all a] the changes in binding bit in values, whereas wh to PDL 45. These ]
TABLE II A2qGES WITH ADVANCING PDL ' ~ S WI KINETICS OF THE RfCHANGES V aibed in the Materials and Methods. Leetins were used at 5 ~g/ml. PhotoF Cells were prepared as described metric readings were relatedd to those of PDL 25 ceils. The results represent at least 10 readings from kM. were calculated from the original readings. triplicate specimens, the S.E.M.
Lectin
PDL 35
PDL 38
PDL 39
PDL 41
PDL 44
Con-A
-10.0 ± 0.6
-13.3 +0.3
-15.3 +0.5
-17.2 _+0.6
-31.5 _+0.4
WGA
-33.1 ± 1.1
-22.2 ± 0.9
-24.8 ± 2.3
-30.4 ± 1.7
-48.4 + 1.4
DBA
+28.2 ± 3.0
+29.5 ± 1.2
+20.0 ± 1.6
+53.5 ± 4.3
+30.4 ± 2.5
0.0
-61.0 ±4.4
-91.2 ±6.8
-33.5 ± 1.6
-29.5 + 1.4
RCA-120
ecrease in the Rf relative to PDL 25 + Positive sign signifiesan increase incre in -Negative sign signifies a decrease the R r relative to PDL 25
ividual cell readings. There is a 4 - 5 e readings on discrete 80 #m 2 areas ( ows that there is a higher level of P 80 ~an 2 cell surface areas than with 1 5elicit as a function of surface are ) L cells, which are larger, have actua
e in cell size cell surfaces at inhibition [ls; however, ~e data may the number
S.
Whole population analyses Analysis of the phenomena at a single cell level wa,s necessar investigate , the use of possible contributions of heterogeneity within cellular populatic low cell concentrations to prevent contact and rather rath, high s mechanical damage make a more analytical population-based assalLy useful. biotin-conjugated lectin assay allows a direct comparison of the data d; and ar msitivity. In addition, the biotin/avidin/enzyme assay allows the clear dist he external surface of the cell because the part of the system re: responsible Lal (enzyme, alkaline phosphatase) is applied in a separate step. In a series of n studies we found that use of the biotinylated lectin and the avidin avidi: at 5 #gt imal results. washing. The biotinydated Incubation times were 90 min each followed by extensive ext sensitivity, and ade(tuate alkaline phosphatase at 5 U/well, or 50 U/ml, provided maxminum m length of time. color development from the PNPP substrate in a reasonable reason~ effe on Table IV presents data from a series of experiments which compare the PDL effect PDL 24 to PDL 32 sho~ lectin binding using the biotin/avidin assay. Cells from fr( show no significant changes in binding except for RCA-120 and DBA. These lectins sh( show a systematic increase in binding between PDL 24 and 32. 3 The DBA binding continu nues to WGA, and RCA-120 sh increase to a maximum at PDL 48. F r o m PDL 32 CON-A, COl how a
T A B L E III ASUREMENT [EMENT OF 80 t~m 2 AREAS OF CELL S U R F A C E CHANGES IN RfAS A MEASUREM
Rf responses from at
rials and least 100 readings. T h e ceUs were prepared as described in the Material de on 80 um 2 areas of cell surface. Methods. Readings were made
Lectin
R filArea
%Rf Change
PDL23
PDL39
CON-A
100.0 -+ 7.4
59.4 -+3.3
-41
WGA
111.4 _+6.2
72.2 _+4.1
-35
DBA
0.6 -+ 0.2
16.3 _+0.7
+2667
111.7 -+7.2
46.5 -+4.1
-59
RCA-120
215
PDL ON THE OD/405 RESPONSE OF I! cultures of IMR-90 ceils at 1 X 105/ml ed in the Materials and Methods.
5-24 h in 96
90 Cultures
32
45
48
CON-A
1.59 -+0.04
1.66 -+0.02
1.42 -+0
1.05 _+0.05
WGA
1.82 -+0.08
1.80 -+0.01
1.35 +0
1.03 + 0.02
DBA
0.22 _+0.07
0.61 -+0.08
0.81 +0
1.39 + 0.09
RCA-120
1.13 _+0.04
1.54 _+0.09
0.94 _+0
0.67 _+0.08
binding level decrease. This corresponds to a change in their gro me when th~ in Fig. 1. These binding changes occur at a point in time
~rphological
as indicated
f the FITCindications of the onset of senescence. These results are simila assay, thus forming a functional link between the single cell and population levels. Table V investigates the effect of cell concentraticon o n the lectin binding resp,)onse. conflu There is a clear response dependence on the level of c,',ellular confluence. At confluency levels from 20% to 80% there is an increase in lectin Ulptake with increased cell ell numbers. num rowth inhibition occurs the ur ~take However, below 20% confluency where low density gr, re of the lectins decreases precipitously to b a c k g r o u n d levels. Above 80% similar results ~ear to occur. These experiments were not possible using the ]FITC-assay system, and appe
TABLE V ONTACT RESP( THE EFFECT OF CELL CONTAC~I ~CT ON THE OD/405 RESPONSE OF IMR-90s OD/405 RESPONSE icate cultures of IMR-90, PDL 24 cells at vaxious concentrations, pre ~repared Mean _+S.E.M. of quadruplicate lated lectins 1 as described in the Materialss and Methods. Confluency was approximated visually. Biotinylated were used at 5 ~g/ml, avidin at 5 ~g/rnl, and biotinylated alkaline phosphatase at 5 U/well. Cell cone. : % Confluency:
2 X 105
I X 10 5
5 X I0 4
2 XlO 4
I XlO 4
80
50
30
20
<20
CON-A
1.50 _+0.13
1.61 _+0.1
1.35 _+0.12
1.10-+0.13
0.15 _+0.02
WGA
0.85 _+0.18
0.73 _+0.1
0.69 _+0.04
0.46 -+0.03
0.08 _+0.02
DBA
0.28 _-/"0.01
0.25 _+0.03
0.24 _+0.03
0.13 _+0.02
0.03 _+0.01
RCA-120
1.00 + 0.14
0.85 _+0.08
0.77 -+0.03
0.65 _+0.03
0,09 _+0.02
Jrface presents oligosaccharide chai DL; (2) low-density growth inhi~
number of (3) contact
ated lectins shows that there is a qua L-dependent r availability of specific mono, or c units on the extemal surface of the IMR-90 cells. These saccharide units are o be part of more complex oligosaccharides, in turn attached to peptide ]; cc r N-linkages. There are significant decreases in CON-A (a-D-Glc, Man), Ma WGA tcNAc), and RCA-120 (b-D-Gal) specificities as well as marked increases )-D-GalNAc) specificities. These quantitative changes were observed observec at the si el and when defined surface areas of individual cells were studied. Thus thes~ ride changes are not apparently affected by the cellular-morphological In ' frequently observed in PDL-dependent systems [ 18]. The biotinylated lectin assay was used to study the bin nenon on a population basis with a higher level of sensitivity than was possi FITC assay. It confirmed the results described above and allowed a detaik )f the lectin binding kinetics as they are related to cell density. There is a cell concentration deper )endent response to the binding of the lectin. This is between 20% and 80% surface confluency conflu and corresponds to the optimal growth conditions for the cell culture. Above 80% confluency contact inhibition slows the cell growth and below b, 20% con fluency an extended exte lag period is observed. We know from studies with tumour tur cells that these data are ar( not an artifact of the assay system because the sensitivity of the assay is sufficient to detect d lectin binding at much lower cell concentrations than that used here (unpublished results). res These data may suggest that the observed saccharide changes ct observed on the cell surface su 9ehavior but also are not only related to cellular senescent behavior alsc to growth regulation in the broader perspective. Furthermore ore, the kinetic studies indicate that the cell surface oligosacch osaccharide changes precede the morrphological expression of senescence and therefore may repr aresent an initiating event. In this his regard the application of the term differentiation seems seem ap)onse. It propriate. The cells' res ponse to a variety of "signals" evokes a differential respon~ may be that one of these ;e responses is a terminal response as has been suggested by Bell ~latory [2], or that this senescence nce behavior is part of a broader more generic growth regul~ mechanism. A number of questions ns are posed by this study. The lectin binding itself is a complex con parameter, therefore alte:~'rations in binding pattern could be the result of the number numb of available sites and/or thete relative affinity of binding within classes of site. The accomac panying study addresses this issue [19]. ACKNOWLEDGEMENTS in The generous support:t of Dr. H.N. Hoppes of Flow Labs is appreciated. This investigation was supported by a grant from the National Institutes of Health (AG00191).
217
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