Báo cáo khoa học: Tumor suppressor p16INK4a ) modulator of glycomic profile and galectin-1 expression to increase susceptibility to carbohydrate-dependent induction of anoikis in pancreatic carcinoma cells ppt

b1-Integrin matur-ation, and transcription of the a5-integrin gene, were enhanced in human SHG44 glioma cells upon down-regulating b4GalT-V expression [39], and the absence of GnT-V in m

and galectin-1 expression to increase susceptibility to

carbohydrate-dependent induction of anoikis in pancreatic carcinoma cells

Sabine Andre´1, Hugo Sanchez-Ruderisch2, Hiroaki Nakagawa3, Malte Buchholz4, Ju¨rgen Kopitz5,Pia Forberich6, Wolfgang Kemmner6, Corina Bo¨ck1, Kisaburo Deguchi3, Katharia M Detjen2,

Bertram Wiedenmann2, Magnus von Knebel Doeberitz5, Thomas M Gress7,

Shin-Ichiro Nishimura3, Stefan Rosewicz2and Hans-Joachim Gabius1

1 Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Germany

2 Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Charite´-Universita¨tsmedizin Berlin, Germany

3 Graduate School of Advanced Life Science, Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Sapporo, Japan

4 Abteilung Innere Medizin I, Universita¨t Ulm, Germany

5 Institut fu¨r Angewandte Tumorbiologie, Klinikum der Ruprecht-Karls-Universita¨t, Heidelberg, Germany

6 Clinic of Surgery and Surgical Oncology, Robert Roessle Hospital, Charite´ Campus Buch, Berlin, Germany

7 Department of Gastroenterology, Endocrinology and Metabolism, University Hospital Giessen and Marburg, Germany

Keywords

anoikis; fibronectin receptor; galectin;

glycosyltransferases; pancreas tumor

Correspondence

S Andre´, Institute of Physiological

Chemistry, Faculty of Veterinary Medicine,

(Received 21 February 2007, revised

23 March 2007, accepted 27 April 2007)

Abbreviations

b4GalT, b1,4-galactosyltransferase; GalNAc, N-acetylgalactosamine; GalNAcT, N-acetylgalactosaminyltransferase; GnT-V,

N-acetylglucosaminyltransferase V; LacNAc, N-acetyllactosamine; MAA, Maackia amurensis; ODS, octadecyl silane; PA, 2-aminopyridine;

pI, isoelectric point; pRb, retinoblastoma tumor-suppressor gene.

Protein glycosylation has functional potential far

beyond the structural roles exerted on the protein

back-bone [1–3] Endowed with a unique high-density coding

capacity, oligosaccharides are versatile biochemical

sig-nals in glycoprotein maturation and routing

intracellu-larly, as well as in diverse cell-surface activities such

as adhesion or trigger mechanisms for signaling to

regulate apoptosis⁄ proliferation [4–7] This paradigm

implies far-reaching functional consequences for the

carefully mapped aberrations of glycosylation upon

malignant transformation [8,9] In global terms,

struc-tural studies on N-glycans from virally transformed

cells (polyoma, Rous sarcoma or hamster sarcoma

virus carrying the v-ras oncogene) have traced distinct,

nonrandom changes in the glycomic profile (i.e an

increased degree of branching and chain extension)

[10–12] Even more important, but still mostly

descrip-tive, oncogene presence has been shown to affect

par-ticular components of the glycosylation machinery, as

documented for H- and N-ras as well as the tyrosine

kinase oncogenes src and her-2⁄ neu [13–16] Signaling

for the measured transcriptional regulation of

N-acetyl-glucosaminyltransferase V (GnT-V) and

a2,6-sialyl-transferase I (ST6Gal-I) is routed through Ras–Raf–

Ets or Ral guanine exchange factors, respectively

[15,16] However, the conclusion to invariably link

the emergence of respective features, especially the

increased b1,6-branching of N-glycans, with the

malig-nant phenotype and therefore with an unfavorable

prognosis in tumor patients, is not justified The

oppos-ite correlation was reported in tumor material from

nonsmall cell lung cancer, neuroblastoma and bladder

cancer [17–19] If it were known which glycoproteins

are key targets, it could become possible to attribute

altered glycosylation to a distinct functionality

In this respect, studies with 3T3 fibroblasts

trans-fected with the SV40 large T antigen gene, HD3 colon

epithelial cells expressing oncogenic ras and human

HT1080 fibrosarcoma cells overexpressing GnT-V

(mentioned above) have illustrated target selection

and, of special note, prominent appearance of the

b1-integrin or the fibronectin receptor (a5b1-integrin)

within this group [20–22] N-Glycosylation of the

fibronectin receptor can be distributed over 14

poten-tial sites in the a5-subunit and over 12 sites in the

b1-chain, covering at least 35 types of oligosaccharides

when analyzed for the protein from human placenta

[23] The processing of these glycan chains is animportant part of integrin maturation, and the glyco-sylation was shown to affect integrin association andclustering, the capacity for fibronectin or lectin bindingand the interaction with the regulatory gangliosides

GT1b and GD3 [21,24–29] These collective insightsshape the hypothesis that remodeling the glycosylation

of the fibronectin receptor can act as a molecularswitch If we could select a cell system in which this in-tegrin plays a major role for the fate of the cells, then

it would be feasible to put our hypothesis to theexperimental test

The recent finding that the tumor suppressorp16INK4a restores susceptibility to anoikis induction inhuman Capan-1 pancreatic carcinoma cells by increas-ing a5b1-integrin expression and surface presentationoffers such a suitable test system [30] We thusassumed that the presence of the tumor suppressor –beyond the transcriptional up-regulation of the a5-inte-grin gene [30] – may engender biologically significantinfluences on glycan synthesis and processing Threelines of evidence support the decision to test our hypo-thesis in this system

First, constitutive p16INK4a expression in humanA549 lung adenocarcinoma cells reduced global b1,4-galactosyltransferase (b4GalT) activity on the cell sur-face by 25%, mainly as a result of reduced expression

of the enzyme b4GalT-I [31] Analysis of transforminggrowth factor b1-induced rapid senescence of thiscell type by northern blots revealed two- to fivefoldincreases in transcription for the b4GalT-II, -III, -Vand -VI genes and abolishment of transcription of theb4GalT-IV gene [32]

Second, comparison of DNA microarray-basedexpression profiles between specimens of pancreaticcancer and normal tissue revealed differential geneactivities, especially up-regulation for b4GalT-V (factor9.91) and b4GalT-I (factor 2.54) and an inverse regula-tion for GnT-IVa and b (factors 3.23 versus )20.27)[33]

Third, the glycosyltransferases b4GalT-V and GnT-Vwere shown to be strongly expressed in a panel of eighthuman cancer lines [34] Transcriptional regulation ofthis b4GalT is under the control of the transcriptionfactor Sp1 (which can activate genes for proteins withpro-growth⁄ survival properties) and probably Ets-1;this property is shared with GnT-V [15,35,36] Gene

orchestrates distinct aspects of glycosylation that are relevant for integrinmaturation and reactivity to an endogenous effector as well as theeffector’s expression This mechanism establishes a new aspect of p16INK4afunctionality

expression of b4GalT-V – and also of b4GalT-I [37] –

can be enhanced by epidermal growth factor and

dom-inant active ras [38] Intriguingly, both enzymes have a

bearing on a5b1-integrin features b1-Integrin

matur-ation, and transcription of the a5-integrin gene, were

enhanced in human SHG44 glioma cells upon

down-regulating b4GalT-V expression [39], and the absence

of GnT-V in murine embryonic fibroblasts led to a

pro-tein kinase C-dependent stimulation of transcription for

the two integrin genes and of clustered cell-surface

pres-entation of the fibronectin receptor [40] In contrast,

human H7721 hepatocarcinoma cells responded to

treatment with GnT-V-specific antisense cDNA with

attenuation of gene expression for both integrins [41]

Obviously, GnT-V-dependent effects in tumors and

cells thus appear to be more difficult to predict than the

consequences of b4GalT-V activity

To delineate any p16INK4a effect on glycosylation at

different levels, we devised a so-far unique, three-step

strategy, starting with cDNA microarray analysis for

glycosyltransferases Because the levels of mRNAs for

these enzymes may or may not directly translate

into the generation of respective oligosaccharides,

we performed global product analysis using 2D

chromatographic profiling The documented evidence

on N-glycan alterations was the reason to focus on this

type of glycosylation In order to find out the

abun-dance of accessible surface glycans, we mapped the

glycomic pattern of native cells using 24 plant lectins,

reactive with N- and⁄ or O-glycans, and then with

human lectins The p16INK4a-positive cells were much

more reactive with galectin-1 than control cells Picking

up this trail, galectin-1 production was found to be

sig-nificantly up-regulated when analyzed by two separate

microarrays, proteomic profiling and flow

cytofluoro-metry, its association with a5b1-integrin was shown and

a positive correlation to anoikis rates was established

The results presented thus reveal a connection between

a tumor suppressor and glycosylation, at the molecular

level probably between a5b1-integrin and galectin-1

This interplay is, at least in part, responsible for

restor-ing susceptibility to anoikis in this cell system

Results

Profiling of gene expression for

glycosyltransferases

In the first set of experiments, we addressed the question

of whether p16INK4a expression in Capan-1 pancreatic

carcinoma cells will have an influence on the expression

of glycosyltransferase genes The sensitivity of detection

was refined to pick up minute signals from this class

of often rather low-abundant mRNA species Toavoid missing important clues, we monitored enzymesinvolved in N- and O-glycan as well as gangliosidebiosynthesis Material from mock-treated and p16INK4a-positive cells was processed under identical conditions,and the ratio of measured signal intensities for cDNApreparations from both types of clones was calculatedfor each enzyme In addition, the average signal inten-sity for the p16INK4a-positive cells served as a relativemeasure of the expression level When setting a thresh-old for a difference in ratio of ± 0.33, a total of 17cases could be compiled; these are detailed in Table 1.The overall mRNA supply for enzymatic capacity

to attach N-acetylgalactosamine (GalNAc) to serine⁄threonine residues of a target protein by a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase(GalNAcT) of the two cell populations was measuredfor the initiation step and ensuing cluster building(here especially GalNAcTs-4⁄ -7) On average, it appeared

to be rather similar The same applied to three testedcases for mucin-type O-glycan extension and itsa2,6-sialylation at the proximal GalNAc moiety byST6GalNAc-IV However, an increased expressionlevel was measured in the cases of mRNA specific fortwo sialyltransferases involved in the synthesis ofa-series gangliosides (Table 1) A high capacity forthe synthesis of a2,3⁄ a2,6-disialyl Lea⁄ Lec epitopes is

an attribute of nonmalignant epithelial cells, reducingthe presence of its sialyl Lea precursor Although thisroute of ganglioside synthesis could favor renormaliza-tion, the tested gene expression profile for core mucin-type O-glycosylation did not reveal any impact ofp16INK4a presence In view of the current literature onepithelial cancer or integrin glycosylation, it is reassur-ing to add that a marked influence on GalNAcT activ-ity could not really be counted upon This situation isdifferent for the branching of N-glycans and elabor-ation of their chain termini

Turning therefore next to enzymes working on plex-type N-glycan structures, no major alteration wasseen in the transcription of GnT-I, -III, -IVB and -Vgenes Because of its importance, the result on GnT-Vwas deliberately ascertained by independent PCRanalysis The same picture emerged in three othergroups of glycosyltransferases: b1,3-galactosyltrans-ferases, b1,3-N-acetylglucosaminyltransferases, exceptfor the type II/V protein (Table 1), and most a-fuco-syltransferases except for a decrease to a ratio of 0.41(signal intensity: 1249) for enzyme VIII introducingthe core-fucose unit, as independently confirmed byreal-time PCR (data not shown) As outlined in theIntroduction, a different situation is anticipated forb4GalTs, and, indeed, the constitutive presence of

com-p16INK4a made its mark on this group Overall, the

most conspicuous changes were detected in the

expres-sion levels of three b4GalT proteins, explicitly

down-regulation of gene expression for proteins I and V and

up-regulation of protein IV Of note, transcriptional

regulation of b4GalTs-I⁄ -V exhibits similarities noted

in the Introduction In terms of signal intensity,

b4GalTs-I⁄ -V were the dominant species Set into

rela-tion with the rather low signal intensities for

b1,3-ga-lactosyltransferases, a preference for type-II termini of

N-glycans is inferred Because the activity of

b4GalT-IV was increased, no drastic decrease in

b1,4-galac-tosylation of glycan chains should occur Owing to

potent galactosylation of the O-glycan core 2

struc-tures by b4GalT-IV, this synthetic route may be

fav-ored A similar trend with up- and down-regulation

within one family was observed for N-glycan-specific

a2,3-sialyltransferases (ST3Gal-III versus -VI) While

the opposite direction of expression levels of these two

a2,3-sialyltransferase genes for enzymes of similar

sub-strate specificity for N-glycans may act in a

compensa-tory manner, the reduction of gene expression of GM3

synthase (ST3Gal-V) may bear upon the capacity for

ganglioside synthesis (Table 1) Looking at O-glycan

a2,3-sialylation, gene expression for ST3Gal-II is

reduced in the p16INK4a-positive cells In contrast, no

modulation is seen for a2,6-sialyltransferase at the

level of mRNA

As with this case, it is essential to note, in generalterms, that the measured extent of gene expressionshould not directly be extrapolated to enzyme and thenproduct presence in a linear manner Of course, whatmatters for the cellular fate is the manifestation of adetected difference at the level of glycan production.Naturally, post-transcriptional regulation and availabil-ity of the activated substrates at the appropriate sitemight also have their share in shifting glycan profiles.Thus, we proceeded to the analysis of actual glycanprofiles via different approaches Based on the presentedresults on significant differences in the display of mRNAlevels of enzymes acting on N-glycans and the documen-ted relevance of N-glycans for integrin maturation, wefirst focused on N-glycans to spot any major differences

in their profile in situ For this purpose, we performed2D chromatographic mapping of neutral N-glycansafter labelling with 2-aminopyridine A major differ-ence in the branching pattern, especially affectingb1,6-branching, will hereby be readily detectable Also,isomers can be separated and molar ratios determined

Chromatographic profiling of N-glycansThe total population of N-glycans was obtained fromcellular extracts, and the reproducibility of results fromseven individual cell batches of the stably transfectedclones was ensured in the first set of experiments Each

Table 1 Microarray data of mRNA expression for glycosyltransferases (± 0.33 from ratio of 1).

Accession Symbol

Ratio p16 ⁄ mock Signal p16 Enzyme functionality NM_003774-0 GALNT4 0.63 670 UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 4 (GalNAcT-4)

NM_017423-0 GALNT7 1.50 2076 UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 7 (GalNAcT-7)

NM_024642-0 GALNT12 0.55 750 UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 12 (GalNAc-T-12)

NM_030965-0 ST6GALNAC5 1.67 1058 GD1a synthase (ST6GalNAc-V); a-series gangliosides

NM_013443-0 ST6GALNAC6 1.77 2122 GD1a synthase (ST6GalNAc-VI); a-series gangliosides, a3,6-disialyl Lecand Leain a-series

gangliosides NM_006577 B3GNT2 0.53 908 b3-N-acetylglucosaminyltransferase 2 (b3GnT-II); initiation and elongation of poly LacNAc NM_032047 B3GNT5 1.65 654 b3-N-acetylglucosaminyltransferase 5 (b3GnT-V); initiation and elongation of poly LacNAc,

O-linked core 3, keratan sulfate, lactotriose NM_001497 B4GALT1 0.33 1642 b4-galactosyltransferase 1 (b4GalT-I); branch extension of N- (b1,2-branch) and O-glycans

(mucin, especially core 4, O-fuc, O-man), lactosylceramide NM_003780 B4GALT2 1.49 506 b4-galactosyltransferase 2 (b4GalT-II); branch extension of N- and O-glycans (see b4Gal-T-I) NM_003778 B4GALT4 4.21 922 b4-galactosyltransferase 4 (b4GalT-IV); poly LacNAc extension on N-glycans (b1,6-branch)

and core 2 O-glycans, neolacto series glycolipids, 6¢-O-sulfated LacNAc NM_004776 B4GALT5 0.29 3680 b4-galactosyltransferase 5 (b4GalT-V); branch extension of N- (b1,6-branch) and O-glycans

(mucin, O-fuc, O-man), lactosylceramide NM_006927 ST3GAL2 0.59 848 CMP-sialic acid:Galb1,3GalNAc a3-sialyltransferase (ST3Gal-II)

NM_006279 ST3GAL3 0.34 1240 CMP-sialic acid:Galb1,3 ⁄ 4GlcNAcb a3-sialyltransferase (ST3Gal-III)

NM_003896 ST3GAL5 0.14 1841 CMP-sialic acid:Galb1,4Glc-Cer a3-sialyltransferase (ST3Gal-V); GM3synthase

NM_006100 ST3GAL6 1.42 890 CMP-sialic acid:Galb1,4GlcNAcb a3-sialyltransferase (ST3Gal-VI); synthesis of

6¢-O-sulfated sLe x

individual peak from the first column was collected

separately, and all oligosaccharides with a molar ratio

above 1% were further analyzed, constituting a total

of 22 different types of neutral N-glycans The elution

properties of 18 N-glycan species are documented to

underlie defined structures As follows, we present the

effect of p16INK4a presence on the molar ratio and the

structures of the major N-glycans

In global terms, the presence of p16INK4a appeared

to shift the balance from complex-type to

oligomanno-syl N-glycans, such as M6.1, M7.1 and M9.1 (Fig 1)

Bi- and tetra-antennary N-glycans surpassed the 5%

threshold of molar ratio in the mock-treated cells

Invariably, the presence of b1,6-branching was reduced

by p16INK4a expression (Fig 1) Only the biantennary

complex-type N-glycan, with both a bisecting GlcNAc

residue and core fucosylation, and the two

type-I-branched triantennary N-glycans without core

fucosylation were slightly more abundant in the

p16INK4a-expressing cells than in the control Of note,

two N-glycans of unknown structure either in the

region of mannose-rich compounds or between the

tri-mannosyl core and the biantennary structure were only

found for the p16INK4a-expressing cells, and no

evi-dence for the emergence of an abundant N-glycan with

poly N-acetyllactosamine extensions could be provided

At this stage, it should be noted that work with whole

cell extracts allows us to reach a statement on total

glycan presence, at the level of sensitivity of this

method Cytoplasmic N-glycans before and after

mat-uration, as well as the cell-surface profile, irrespective

of accessibility, will be simultaneously evaluated How

the pattern of glycans presented on the cell surface

and accessible for binding partners looks will have to

be clarified by a different method Binding studies with

glycan epitope-specific probes conducted on intact cellsare suited to address this question For this purpose,forming a panel of plant lectins is a validatedapproach Although they will not monitor any glycosy-lation-dependent change of conformation or functionalstatus of a glycoprotein, the glycan(s) in these casesdirectly acting on its (their) protein backbone, system-atic application of these sensors for glycan structures is

a step to define potential in situ effectors on the glycanside In order to cover the main classes of glycan con-stituents we selected 24 plant agglutinins The list ofproteins and their sugar specificities are presented inTable 2

Profiling of cell-surface glycans by plant lectins

In the first step of these experiments, we establishedthe concentration and sugar dependence of lectinbinding, as illustrated in the supplementary material(Fig S1) These experiments were also instrumental indetermining a common concentration to detect relativedifferences by comparative mapping and to avoid anytoxic effects of lectins The experimental series wassystematically performed in parallel under identicalconditions for the two cell populations Hereby, anyparameter change by prolonged or differential cultureperiods was avoided For convenient comparison, wemeasured the percentage of positive cells and meanfluorescence intensity in each panel, as listed in Fig 2

In full accordance with the results on the abundance

of mRNA for the GalNAcTs, the presence of GalNAcresidues, measured with five different lectins, wasrather similar except for VVA (Fig 2) This lectin mayreact preferentially with globo- and isoglobotetraosyl-ceramides and not mucin-type O-glycans An apparent

Fig 1 Quantitative profiling of N-glycans Complete representation of the N-glycan profiles of mock- and p16INK4a-transfected pancreatic cinoma cells and their molar ratios determined by the 2D mapping technique The N-glycan structure is given for each case, and an inset is added for explanation.

car-preference for the Thomsen-Friedenreich epitope

anti-gen was detected for p16INK4a-positive cells by PNA

and jacalin This result may reflect either elevated core 1

synthesis or efficient core 1 masking by a2,3-sialylation

in the mock control, as suggested by the microarray

data It was therefore essential to study this issue in

greater detail (please see the paragraph below) Using

the standard concentration Maackia amurensis-II

(MAA-II), the percentage of positive cells was

enhanced for mock-treated clones, possibly attributable

to different gene expression levels for the enzymes

responsible for the two sialylation steps

N-Glycosylation, especially with core fucosylation,appeared to be accessible on the cell surface to ahigher extent in the p16INK4a- versus mock-transfectedcell populations Glycan profiling had revealed anincrease for biantennary glycans of this type containingthe additional bisecting GlcNAc unit, in contrast to anotherwise decreased level of core fucosylation (Fig 1),

in full accord with array data of a-fucosyltransferaseVIII Testing two probes with similar specificities(i.e LCA⁄ PSA) served as an internal control Appar-ently, the chromatographic profiling, on average,detected more substituted N-glycan in mock controls

Table 2 Lectin panel for glycan profiling of cell surfaces listed in alphabetic order.

Monosaccharide specificity Potent oligosaccharideaArtocarpus integrifolia (jack fruit) Jacalin (JAC) Gal ⁄ GalNAc Galb3GalNAca

Canavalia ensiformis (jack bean) Con A Man ⁄ Glc GlcNAcb2Mana6(GlcNAcb2Mana3)Manb4GlcNAc

Datura stramonium (thorn apple) DSA GlcNAc (GlcNAc)n, Galb4GlcNAcb6(Galb4GlcNAcb2)Man (Galb4GlcNAc)3

Griffonia simplicifolia II GSA II GlcNAc GlcNAcb4GlcNAc, N-glycans with terminal, nonreducing-end

GlcNAc Lens culinaris (lentil) LCA Man ⁄ Glc N-glycan binding enhanced by core-fucosylation

Lycopersicon esculentum (tomato) LEA b core and stem regions of high-mannose-type N-glycans,

(GlcNAcb3Galb4GlcNAcb3Gal)nof complex-type N-glycans Maackia amurensis I (leukoagglutinin) MAA I b Neu5Aca3Galb4GlcNAc ⁄ Glc

Maackia amurensis II (haemagglutinin) MAA II b Neu5Aca3Galb3(a6Neu5Ac)GalNAc

Phaseolus vulgaris erythroagglutinin

(kidney bean)

(GlcNAcb2-Mana3)(GlcNAcb4)Manb4GlcNAc Phaseolus vulgaris leukoagglutinin

(kidney bean)

PHA-L b Tetra- and triantennary N-glycans with b6-branching Pisum sativum (garden pea) PSA Man ⁄ Glc N-glycan binding enhanced by core-fucosylation

Sambucus nigra (elderberry) SNA Gal ⁄ GalNAc Neu5Aca6Gal ⁄ GalNAc

Solanum tuberosum (potato) STA b (GlcNAc)nwith preference for high-mannose-type N-glycans

Triticum vulgare (wheat germ) WGA GlcNAc ⁄ Neu5Ac (GlcNAc) n , Galb4GlcNAcb6Gal

Viscum album (mistletoe) VAA Gal Galb2(3)Gal, Gala3(4)Gal, Galb3(4)GlcNAc without ⁄ with

a2,6-sialylation, Fuca2Gal

a Based on previously compiled information [121], extended and modified; b no monosaccharide known as ligand.

Fig 2 Profiling of cell-surface glycans by plant lectins Semilogarithmic representation of fluorescent surface staining by biotinylated plant lectins (for an explanation of the acronyms and listing of oligosaccharide specificity, please see Table 2) of mock-transfected (gray line) and p16 INK4a -transfected (black line) Capan-1 pancreatic carcinoma cells determined in parallel assays Quantitative data on the percentage of positive cells and fluorescence intensity are given in each panel (first line: mock-treated cells; second line: p16 INK4a -transfected cells) The concentration of the biotinylated lectins was 0.5 lgÆmL)1except for SNA and DBA (1 lgÆmL)1), SJA and WGA (2 lgÆmL)1) and MAA-I (5 lgÆmL)1).

when compared with the relatively increased lectin

reactivity on the cell surface for p16INK4a-expressing

cells, indicating disparities in the levels of accessibility

and ligand preferences The chromatographic profiling

and cell-surface detection of N-glycans with bisecting

GlcNAc by PHA-E could easily be reconciled, product

formation substantiating efficiency of only minute

quantities of detectable mRNA for GnT-III, whereas no

major accessibility difference could be discerned

regard-ing lectin bindregard-ing of the b1,6-branch by PHA-L (Fig 2)

Due to the potential interference by a2,6-sialylation,

this result, as noted for the Thomsen-Friedenreich

antigen epitope and a2,3-sialylation above, had to be

further scrutinized (please see below for the effect of

sialidase treatment) A clear difference in lectin binding

concerned the presence of accessible GlcNAc moieties

measured by applying DSA⁄ WGA but not seen to this

extent with GSA-II and STA Expression changes in

the b4GalT family may underlie this staining property

LEA, a marker for extensions of N-glycan branches by

N-acetyllactosamine (LacNAc) units, failed to provide

clear evidence for marked cell-surface differences,

arguing in favour of the concept of compensatory

change within the tested

b1,3-N-acetylglucosaminyl-transferases and b4GalTs, as also seen in

chromato-graphic profiling Because similar cell staining was

measured with GNA, the contribution of the dual

reactivity of LEA to high-mannose-type N-glycans will

probably have no influence on this result Similarly,

the abundance of accessible poly N-acetyllactosamine

chains was rather similar, prompting a final check of

chain-end galactosylation

The two respective probes (VAA and ECA) revealed

more intense staining of the p16INK4a-reconstituted

clones than of the mock control (Fig 2; supplementary

Fig S1) This result does not simply reflect the

micro-array data when adding up signal intensities for

b4GalT-specific cDNAs As depicted above, sialylation

will make its presence felt in this approach Because it

can mask terminal galactose residues for lectins,

meas-urement of its status was essential a2,3-Sialylation of

N-glycans monitored comparatively with MAA-I at a

fairly high concentration showed a slight preference

for the p16INK4a-positive clone, indicating a

compensa-tory balance for ST3Gal-III⁄ -IV ⁄ -VI gene expression

levels By contrast, cell positivity expressed as cell

percentage was lowered in these cells when measuring

lectin-reactive N-glycan-specific a2,6-sialylation, despite

similar extents of ST6Gal-I gene expression (Fig 2)

Ectopic ST6Gal-I expression in the p16INK4a-positive

cells did not change this parameter (data not shown)

As mentioned above, this observation on the

noniden-tical degree of sialylation makes it mandatory to

deter-mine comparatively the impact of sialylation on thebinding of plant lectins sensitive to its presence Inaddition to the inhibition control with haptenic sugar,the pre-exposure of cells to neuraminidase is, at thesame time, a second control for ensuring carbohy-drate-dependent binding of SNA Standard conditionsfor enzymatic treatment were established, minimizingthe influence on the cell phenotype In line with theinhibition studies, enzymatic pretreatment significantlyreduced (mock control) or almost completely abolished(p16INK4a-positive cells) SNA binding (Fig 3) Thesame effect was observed for MAA-II used at a nearlysaturating concentration of 5 lgÆmL)1 (Fig 3) Inaddition to its control character, these results supportthe evidence for a quantitative difference in thea2,6-sialylation status between the two cell popu-lations Should the level of mucin-type O-glycana2,3-sialylation also be lowered in the p16INK4a-posit-ive cells, as suggested by the microarray data, thenneuraminidase activity should enhance PNA staining

of the control cells, with a minor influence on thep16INK4a-expressing cells and on DBA staining Theimportance of this aspect has been pointed out above.Fittingly, PNA, but not DBA, positivity was markedlyimproved by the enzymatic removal of sialic acid resi-dues from the cell surface for mock-transfected cellsbut not for the p16INK4a-positive cells (Fig 3) Theminor effect on the p16INK4a-positive cells probablyindicates the presence of mucin-type core 2 tetrasaccha-rides, favored by an increased b4GalT-IV presenceand reduced O-glycan a2,3-sialylation Thus, lectin-accessible mucin-type O-glycosylation appeared to

be rather equally abundant, but the levels of itsa2,3-sialylation were definitely different The apparentpreference for mock-treated cells to carry O-glycan core

1 a2,3-sialylation can be accounted for – at least in part– by the microarray data

Because the presence of a2,6-linked sialic acids canimpede PHA-L binding, the same procedure was alsocarried out in this case Using a lectin concentration of

1 lgÆmL)1, increased staining was seen in both cellpopulations, and accessibility was still at an increasedlevel for the p16INK4a-positive cells (Fig 3) To avoidunderestimation of the presence of b1,6-branchedN-glycans in the mock control, the remarkably differ-ent levels of SNA binding after standard neuramini-dase treatment must be recalled In essence, data fromchromatographic mapping square well with the lectinprofiling A major result emerging from these experi-ments is that the extents of sialylation of N- andmucin-type O-glycans in the two cell populations(a2,6-substitution for N-glycans, a2,3-modification forO-glycans) are different In functional terms, these

processes may generate or mask sites for contact in situ

with endogenous lectins As the results with the

b-gal-actoside-specific lectins VAA⁄ ECA revealed, the levels

of accessible galactose residues were remarkably

differ-ent between the two cell populations Monitoring of

cell-surface binding by plant lectins thus pinpointed

disparities in accessible glycans These observations

directed our interest to the detection of endogenous

lectins They might turn these newly defined properties

on the level of cell-surface glycosylation into effects, in

this case on the level of susceptibility to anoikis

With focus on sialylation⁄ galactosylation, the main

groups of human lectins that can read and translate

such differences are the C-type lectins, siglecs and

galectins [42] The ensuing microarray monitoring of

expression of 42 C-type lectins and siglecs-2, -3, -5, -6,

-7 and -9 failed to provide positive data or, if positive,

a difference in signal intensities When testing the third

mentioned lectin family, indications for transcription

of galectin genes were collected As further ascertained

by systematic RT-PCR analysis within this lectin

fam-ily, transcription of genes for galectins-1, -3, -7 and -9,

respectively, was detected, the most pronounced signal

(i.e 5369) seen in the case of galectin-1 in p16INK4a

-positive cells (data not shown) Having herewith

provided evidence for gene expression of members of

this galactoside-specific lectin family, we next probed

whether human adhesion⁄ growth-regulatory galectins

can bind to the cells, as shown for the

galactoside-specific lectins ECA⁄ VAA By testing more proteinsthan just galectin-1, the individual binding properties

of the structurally closely related members of this ily can also be profiled in one cell system, a compar-ison so far not reported For this purpose, we purifiedthe lectins and then biotinylated them under activity-preserving conditions, ascertained a lack of harmfuleffects on sugar binding by activity assays and deter-mined the labeling efficiency with 2–8 modified resi-dues per carbohydrate-recognition domain As in thebinding studies with plant lectins, we routinely per-formed experiments to assess concentration depend-ence and inhibition of galectin binding by haptenicsugar

fam-Profiling of galectin bindingThe measurements with the two galactoside-specificplant lectins and also with MAA-I (galectins toleratea2,3- but not terminal a2,6-sialylation) led to theexpectation that these human lectins may preferentiallybind to p16INK4a-expressing cells with their increasedpresence of these epitopes Indeed, the respectivestudies confirmed this notion already in the first set ofexperiments with galectin-1, when documenting thedependence of cell staining on lectin concentration and

on glycan binding (supplementary Fig S2, first andsecond panels) This homodimeric family member is apotent cross-linker for glycans on the cell surface

Fig 3 Effect of sialidase treatment on the cell-surface binding of plant lectins Semilogarithmic representation of fluorescent surface staining

of mock-transfected (Mock) and p16 INK4a -transfected (p16) Capan-1 pancreatic carcinoma cells without the incubation step using the ated lectin (shaded) and after incubation with the labeled probe (0.5 lgÆmL)1of PNA, 1 lgÆmL)1of PHA-L, 5 lgÆmL)1of SNA and MAA-II, as well as 10 lgÆmL)1of DBA), without (gray line) or after (black line) sialidase treatment Quantitative data are presented as defined in the legend to Fig 2.

biotinyl-Besides using haptenic sugar to relate lectin activity to

binding, we tested two mutants of human galectin-1

Their carbohydrate-binding activity was impaired by

a crucial substitution (W68L, E71Q) in the

carbo-hydrate-recognition domain The loss of binding to

the p16INK4a-transfected cells compared with the

His-tagged wild-type control protein served as an

independent validation of the inhibition control

(sup-plementary Fig S2, third panel) The

concentration-and carbohydrate-dependent binding is also illustrated

for the chimera-type galectin-3 In this case, it is

obvious that the cells of the mock control are also

rather reactive, when considering cell-percentage

posi-tivity (supplementary Fig S2) Given this indication

for intergalectin differences, we systematically assayed

a series of human galectins to define staining

proper-ties with these human effector proteins As shown in

the supplementary material (Fig S3), there is a clear

trend for galectin reactivity correlating with tumor

suppressor presence The tandem-repeat-type galectin-8

reacted similarly to galectin-9 (data not shown) The

most prominent change for the combination of both

quantitative cell staining parameters was seen in the case

of galectin-1 Glycan-dependent galectin binding to

these cells is thus detectable, it is not a uniform

characteristic, and, finally and even more importantly,

the conspicuous difference of galectin-1 binding to the

two cell populations gives further study a clear

direction

As a result of the blocking effect of terminal

a2,6-sialylation on galectin-1 binding, we assumed that

this type of sialylation will mask galectin-1-reactive

sites on the cells of the mock control If therefore

exposed to a sialidase, these cells should become

react-ive, as shown for SNA or PNA binding in Fig 3

Indeed, reduction of sialylation under standard

condi-tions increased cell binding markedly for the mock

control, whereas the p16INK4a-transfected cells showed

only slightly improved binding properties (Fig 4)

Galectin-1 specificity renders it very likely that

remov-ing the blockremov-ing a2,6-sialylation underlies this

param-eter change That the same reactivity pattern was seen

for galectin-3 constitutes not only an inherent control

As galectin-3 tolerates a2,6-sialylation in poly

N-acetyllactosamine chains already at the level of the

dimer, in stark contrast to galectin-1 [43], these results

signified no notable difference for the presence of such

chain extensions between the cell types, in full

agree-ment with LEA and microassay data In view of an

effector function, the differential status of sialylation

thus appeared to influence the accessibility to ligand

sites effectively This result might become functionally

relevant if a functional relationship between galectin-1

and the expression of the p16INK4a protein could bedelineated In this sense, the p16INK4a-dependentincrease of the presentation of binding sites by reduceda2,6-sialylation might even be associated withenhanced galectin-1 expression, this regulatory eventaccomplishing optimal sensitivity We put this reason-ing to the test in a stepwise manner by a gene array,

by northern blotting⁄ nuclear run-off experiments, byproteomic profiling and by flow cytofluorometry

Identification of up-regulation of galectin-1expression

Quantitative determination of the presence of 1-specific mRNA indicated a p16INK4a-associatedincrease In detail, we comparatively probed 1996cDNAs in an array designed for pancreas tissue andits cancer development and applied stringent criteriafor defining up-regulation The threshold of 50%increase was surpassed by 16 signals Galectin-1 genetranscription was the most prominent defined case

galectin-at a p16INK4a⁄ mock ratio of 3.01 (supplementary

Fig 4 Effect of sialidase treatment on the cell-surface binding of human galectins Semilogarithmic representation of fluorescent staining of mock-transfected (Mock) and p16INK4a-transfected (p16) Capan-1 pancreatic carcinoma cells without the incubation step using the biotinylated lectin (shaded) and after incubation with labe- led galectins-1 and -3 (gal-1 and gal-3 used at 10 lgÆmL)1) without (gray line) or after (black line) sialidase treatment Quantitative data are presented as defined in the legend to Fig 2.

Table S1) Northern blotting and nuclear run-off

experiments confirmed the array data and

substan-tiated an increase in de novo transcription (data not

shown) Extending our work from the mRNA level,

we next performed a proteomic analysis with the

inten-tion of establishing whether the galectin-1 protein is

produced at an amount reflecting gene expression

With a total of 600–670 spots per gel and a

reproduci-bility of spot assignment between different gels of

89.4–99%, we detected one spot among the 48 signals

that showed a consistent increase in staining by 50%

in p16INK4a-positive cells, relative to control cells, with

mass⁄ isoelectric point (pI) characteristics compatible

with galectin-1 As shown in Fig 5, we confirmed this

hypothesis by western blotting and mass-spectrometric

fingerprinting On the basis of staining of protein by a

dye or the western blotting procedure, galectin-1

pres-ence as protein was found to be significantly

up-regula-ted (P¼ 0.0154, P ¼ 0.0027) The advantage of the

proteomic profiling compared with western blotting

after 1D electrophoresis, shown separately in Fig 6A

as a control, is the exclusion of formation of any

galectin-1 variants based on different pI values After

synthesis, the protein underwent secretion, because we

detected its presence in the medium (data not shown)

As a consequence it may then associate with the cell

surface, prompting flow-cytofluorometric analysis

Applying the antigalectin-1-specific immunoglobulin

for cell-surface detection, the difference in protein

pro-duction translated into increased surface presence in

the p16INK4a-positive cells (Fig 5) We deliberately

tes-ted several cell batches and consistently measured an

enhanced cell-surface presentation in the p16INK4a

-pos-itive cells under standard culture conditions When

determining the level of inhibition of galectin-1 binding

by the haptenic sugar, lactose, a notable difference

became apparent It was comparatively lower in this

cell type than in the control cells, a measure for strong

affinity of the endogenous lectin to a set of particular

surface glycans In fact, endogenous galectin-1 could

hardly be stripped off the cell surface, even in the

pres-ence of 200 mm lactose In comparison, lectin binding

from the medium as source was much more sensitive

to inhibitor presence, as observed from loading the

cells with galectin-1 up to saturation (supplementary

Fig S2), intimating visualization of the gradient of

decreasing affinity for binding multivalent ligands seen

in a recent model study [44] To exclude that the

p16INK4a-dependent up-regulation of galectin-1 is a

singular event confined to Capan-1 cells only, we

tes-ted Dan-G pancreatic cancer cells without and with

p16INK4a expression Of note, these two cell lines give

insight into the specificity of the effect as a result of

their differences in the status of the retinoblastomatumor-suppressor gene, pRb Increased galectin-1expression was determined by western blotting in bothclones of engineered transfectants with p16INK4aexpres-sion, despite maintained pRb status (data not shown) It

is thus tempting to propose a functional correlationbetween the detected galectin-1 up-regulation, theincreased presentation of galectin-1-binding sites inp16INK4a-expressing cells and acquisition of anoikis sus-ceptibility associated with the fibronectin receptor

Induction of anoikis by galectin-1

In order to test the hypothesis described above, we sued two independent approaches First, we establishedstable clones with reduced galectin-1 production bytransfection with a vector harboring full-length cDNAfor galectin-1 in the antisense orientation After con-firming the reduction of galectin-1 presence, to a levelcharacteristic of wild-type cells, by western blotting,the cells of such a clone were subjected to monitoringlevels of anoikis In line with our hypothesis, the extent

pur-of anoikis was correlated to the level pur-of galectin-1 sent (Fig 6A) Second, we forced an increase of galec-tin-1 production on wild-type cells weakly positive forgalectin-1 binding, using proliferating cell nuclear anti-gen as an internal control Anoikis induction wasenhanced, even showing a trend for dose dependenceamong the tested clones (Fig 6B) Corroborating thisbiological effect on wild-type cells artificially overex-pressing galectin-1 we could, in parallel, elicit anoikis

pre-in regular wild-type cells by addpre-ing the lectpre-in to ium at a concentration of 125 lgÆmL)1 (data notshown) The presence of haptenic sugar interfered withthis process, revealing carbohydrate dependence, as didthe presence of the predominantly monomeric galectin-

med-3, revealing a requirement for cross-linking (data notshown) Finally, to connect galectin-1 with thep16INK4a-associated increase of cell-surface presence ofthe fibronectin receptor, we reasoned that preparations

of the integrin, when immunoprecipitated fromp16INK4a-positive cells, should contain galectin-1.Using cells grown adherent or in suspension, we testedthis assumption Western blotting revealed that galec-tin-1 was indeed co-immunoprecipitated with thisglycoprotein, the levels of galectin-1 presence beingconsistently higher in p16INK4a-positive cells than incontrol cells (Fig 6C) Independently, the antibodyagainst the a5-subunit was effective at markedly redu-cing the extent of binding of labeled galectin-1 to thecell surface (data not shown) These results imply thatthe a5b1-integrin is a binding partner of anoikis-indu-cing galectin-1

Fig 5 Enhanced galectin-1 expression in p16INK4a-reconstituted cells Aliquots of total protein (200 lg) from mock-transfected (Mock) and p16 INK4a -transfected (p16) Capan-1 pancreatic carcinoma cells were subjected to 2D gel electrophoretic separation and silver staining The kDa-section where galectin-1 presence can be expected was marked (A, B) and the putative position of galectin-1 was labeled Western blot (WB) analysis with equal quantities of protein and 1 lgÆmL)1of galectin-1 antibody as probe revealed spots at this position after antigen visualization by enhanced chemiluminescence (top panel) Mass-spectrometric fingerprinting after digestion of the protein of this spot by trypsin ascertained identity to galectin-1, and quantification of the staining intensity in gel electrophoretic (2-DE) and WB analyses revealed statistically significant up-regulation (middle panel) Cell-surface detection of galectin-1 in flow-cytofluorometric analysis was performed using

20 lgÆmL)1of polyclonal galectin-1 antibody as probe and fluorescent goat anti-rabbit IgG as the second-step reagent (bottom panel).