Báo cáo khoa học: Tumor suppressor p16INK4a: Downregulation of galectin-3, an endogenous competitor of the pro-anoikis effector galectin-1, in a pancreatic carcinoma model pptx

Results Expression of p16INK4adecreases the level of Gal-3 In order to delineate any impact of the tumor suppres-sor p16INK4a on Gal-3 expression we worked with Capan-1 pancreatic carcin

an endogenous competitor of the pro-anoikis effector

galectin-1, in a pancreatic carcinoma model

Hugo Sanchez-Ruderisch1,*, Christian Fischer1, Katharina M Detjen1, Martina Welzel1,

Anja Wimmel1, Joachim C Manning2, Sabine Andre´2and Hans-Joachim Gabius2

1 Medizinische Klinik m.S Hepatologie und Gastroenterologie, Charite´-Universita¨tsmedizin Berlin, Germany

2 Institut fu¨r Physiologische Chemie, Ludwig-Maximilians-Universita¨t Mu¨nchen, Germany

Introduction

The tumor suppressor p16INK4a, a frequent target for

deletion mutations underlying carcinogenesis, is known

as a binding partner of cyclin-dependent kinases

CDK4 and CDK6, interfering with their association

with D-type cyclins [1,2] Emerging evidence broadens

the spectrum of p16INK4a functionality beyond

cell-cycle control In fact, recent experiments have

unrav-eled an effect on distinct aspects of gene expression

One functional consequence is to restore the

suscepti-bility of tumor cells to anoikis (the category of

apopto-sis caused by inadequate or inappropriate cell–matrix

contacts) In detail, enhanced production of the

a5-integrin subunit and also an increased cell-surface presence of a5b1-integrin (the fibronectin receptor) were detected in p16INK4a-restituted Capan-1 pancre-atic carcinoma cells [3] Routing of this glycoprotein and the levels of its cell-surface presentation, binding activity and capacity for downstream signaling may, in principle, depend not only on the protein part, but also on its glycosylation Because the fibronectin recep-tor is heavily glycosylated with 26 sites for N-glycosyl-ation [4] and variN-glycosyl-ations in the structures of glycan

Keywords

anoikis; galectin; glycosylation; integrin;

pancreatic carcinoma

Correspondence

K M Detjen, Charite´-Universita¨tsmedizin

Berlin, Campus Virchow-Klinikum, Med.

Klinik m.S Hepatologie und

Gastroenterologie, Augustenburger Platz 1,

D-13353 Berlin, Germany

Fax: +49 30 450 559939

Tel: +49 30 450 559679

E-mail: katharina.detjen@charite.de

*Present address

Center for Cardiovascular Research,

Charite´-Universita¨tsmedizin Berlin, Germany

(Received 20 May 2010, revised 1 July

2010, accepted 5 July 2010)

doi:10.1111/j.1742-4658.2010.07764.x

The tumor suppressor p16INK4a has functions beyond cell-cycle control via cyclin-dependent kinases A coordinated remodeling of N- and O-glycosyla-tion, and an increase in the presentation of the endogenous lectin

galectin-1 sensing these changes on the surface of pgalectin-16INK4a-expressing pancreatic carcinoma cells (Capan-1), lead to potent pro-anoikis signals We show that the p16INK4a-dependent impact on growth-regulatory lectins is not lim-ited to galectin-1, but also concerns galectin-3 By monitoring its expression

in relation to p16INK4a status, as well as running anoikis assays with galec-tin-3 and cell transfectants with up- or downregulated lectin expression, a negative correlation between anoikis and the presence of this lectin was established Nuclear run-off and northern blotting experiments revealed an effect of the presence of p16INK4a on steady-state levels of galectin-3-spe-cific mRNA that differed from decreasing the transcriptional rate On the cell surface, galectin-3 interferes with galectin-1, which initiates signaling toward its pro-anoikis activity via caspase-8 activation The detected oppo-site effects of p16INK4a at the levels of growth-regulatory galectins-1 and -3 shift the status markedly towards the galectin-1-dependent pro-anoikis activity A previously undescribed orchestrated fine-tuning of this effector system by a tumor suppressor is discovered

Abbreviations

Gal-1, galectin-1; Gal-3, galectin-3; PCNA, proliferating cell nuclear antigen; poly-HEMA, poly(2-hydroxyethyl methacrylate).

chains, such as status of sialylation, have a bearing on

protein functions in general and on integrins in

partic-ular [5–8], glycan remodeling affords an attractive level

of regulation of integrin functionality The assumption

of a modulatory impact of p16INK4aat the level of

gly-cosylation was a reasonable and testable hypothesis

This hypothesis was verified by combining glycogene

microarray analysis, chromatographic glycan profiling

and lectin binding [9] Influencing N- and O-glycan

galactosylation and sialylation, salient biochemical

sig-nals in protein–carbohydrate⁄ protein interplay [10–12],

have thus become a new aspect of p16INK4a

functional-ity Hereby, the tumor suppressor can affect integrin

processing and routing, as well as its affinity for

protein ligands

In addition, modified glycan chains can be engaged

in recognitive interactions at the cell surface with

endogenous lectins, which translate sugar-encoded

messages into cellular responses [13–15] Even

seem-ingly small modifications can act as potent switches of

affinity In fact, altering branch-end positions, the

sta-tus of core substitutions and glycan density strongly

affects lectin reactivity, as measured for example for

members of the family of adhesion⁄ growth-regulatory

galectins [16–19] Fittingly for a functional implication,

microarray analysis on a set of 1996 cancer-associated

genes, proteomic profiling and cytofluorometry and

anoikis assays revealed the upregulation of

homo-dimeric galectin-1 (Gal-1) as a carbohydrate-binding

effector for anoikis induction under the control of

p16INK4a [9] Thus, this tumor suppressor sensitizes

cells for the onset of anoikis by increasing the

comple-mentary sides of pro-anoikis protein–carbohydrate

interactions

As the term Gal-1 implies, the functionality of this

protein might be embedded in a network with other

family members [20] This raises the possibility of

addi-tive or antagonistic effects Exploring this issue, model

studies on SK-N-MC neuroblastoma cells illustrated

the potential of other galectins, especially galectin-3

(Gal-3), to factor in growth inhibition by Gal-1 at the

level of cell-surface ligand binding [21,22] Having

recently delineated a direct connection between

p16INK4a and Gal-1 [9], the question arises as to

whether the influence of p16INK4a is restricted to this

family member The following four lines of evidence

direct the focus of attention on Gal-3 First, it is

func-tionally antagonistic to Gal-1 in the neuroblastoma

system by blocking access to ganglioside GM1, the

common galectin ligand on the cell surface in these

cells [21] Second, activating K-ras mutations are

com-mon in pancreatic cancer, and Gal-3 (but not Gal-1)

interacts with oncogenic K-ras–GTP to promote Raf-1

and phosphoinositide 3-kinase activities, as well as a signal attenuating extracellular signal-regulated kinase [23] This is in accordance with the lack of aberrantly enhanced extracellular signal-regulated kinase signaling

in pancreatic tumors which harbor the mentioned gene defect [24] Third, Gal-3 protects BT549 breast cancer cells from anoikis [25] Fourth, Capan-1 wild-type cells are known to express Gal-3 [26] On this basis, we investigated the influence of the presence of p16INK4a

on the level of Gal-3 production and possible func-tional competition between galectins-1 and -3 The reported results document the relevance of Gal-3 in interfering with anoikis induction and, more impor-tantly, shed light on the intriguing capacity of this tumor suppressor to coordinately exploit this aspect of the galectin network

Results

Expression of p16INK4adecreases the level of Gal-3

In order to delineate any impact of the tumor suppres-sor p16INK4a on Gal-3 expression we worked with Capan-1 pancreatic carcinoma wild-type and vector-transfected cells (mock) as well as three independently generated clones stably transfected with p16INK4atumor suppressor cDNA (p16 1-3) Of note, these clones had been studied previously, revealing increased expression and cell-surface presentation of the a5-integrin subunit and Gal-1 [3,9] Western blot analyses first resulted in the expected pattern for the p16INK4aprotein, then con-firmed the reported presence of Gal-3 in Capan-1 wild-type cells [26] and excluded an effect of the control transfection on Gal-3 and general protein synthesis (Fig 1A) In contrast to the mock process, the presence

of p16INK4a protein had an effect A clear decrease in the intensity of signals for Gal-3 was invariably observed, although proliferating cell nuclear antigen (PCNA) staining as a control for loading remained rather constant (Fig 1A,B) To examine whether such a negative correlation between Gal-3 and p16INK4a can also be seen in vivo, we processed routinely fixed sec-tions from normal pancreas tissue and pancreatic can-cer immunohistochemically The resulting staining profiles, shown in Fig S1, confirmed an inverse expres-sion pattern in accordance with the in vitro data In principle, Gal-3 can act as an anti-anoikis effector in the cell and⁄ or at the cell surface, for example, blocking Gal-1 functionally as seen previously in a neuroblas-toma model [21] In view of the proven pro-anoikis cell-surface activity of Gal-1, the cultured cells provided the opportunity to test the hypothesis of reduced

cell-surface expression of Gal-3 Indeed, cytofluorimet-ric monitoring of mock-transfected and p16INK4a -resti-tuted Capan-1 cells revealed that cell-surface Gal-3 was downregulated (Fig 1C) As reported previously [9], Gal-1 cell-surface presentation determined in the con-trol increased (not shown) The presence of the tumor suppressor thus reduced the level of Gal-3 and its cell-surface presentation To further test whether Gal-3 pro-tein abundance was regulated by prolonged culture in suspension, we maintained cells for up to 24 h under this condition, with the percentage of cells undergoing anoikis increasing as expected in p16INK4a-expresssing cells (Fig 2A) Loss of anchorage did not notably change the level of Gal-3 detected in western blots (Fig 2B) These results document a significant decrease

in the presence of Gal-3 in tumor-suppressor-positive cells, which is not further enhanced in suspension cul-ture This suggests regulation at the level of transcrip-tion, as previously detected for Gal-1 [9] To test this,

we determined the steady-state mRNA concentration

by northern blotting and the de novo transcription rate

by run-off assays

p16INK4anegatively affects Gal-3 mRNA availability

Northern blotting using samples from p16INK4a-positive cells revealed a conspicuous decrease in the availability

of Gal-3-specific mRNA, when compared with mock-transfected controls, whereas similar mRNA levels for the housekeeping gene GAPDH were present irrespec-tive of cell status (Fig 2C, left) This diminished steady-state amount may be because of alterations in de novo production or availability Nuclear run-off assays, which report on de novo synthesis, revealed rather simi-lar signal intensities in samples from mock- and p16INK4a-transfected cells for the two tested housekeep-ing genes and for Gal-3 (Fig 2C, right) De novo Gal-1 gene transcription, by contrast, increased significantly in the presence of the tumor suppressor (not shown) Thus, the transcriptional rate is a major factor in increasing the production of Gal-1, whereas post-transcriptional processes decrease Gal-3 production We propose a functional relevance for this alteration In order to prove an effect of Gal-3 on anoikis in this cell system, especially regarding a functional antagonism with Gal-1

at the level of the cell surface, we followed three routes

of investigation to test the validity of this assumption

Gal-3 is an inhibitor of anoikis

In the first set of experiments, we tested the capacity

of Gal-3 to block anoikis in p16INK4a-expressing cells

10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4

A

B

C

Fig 1 p16 INK4a restitution inhibits Gal-3 expression (A) Western

blot after 1D SDS ⁄ PAGE [15% gel, proteins blotted onto

poly(vinyli-dene fluoride)] with protein extracts from Capan-1 wild-type (wt),

mock-transfected and three p16 INK4a -expressing clones incubated

with antibodies to p16INK4a, Gal-3 and PCNA, respectively The level

of Gal-3 is reduced in p16 INK4a -expressing clones (B) Western blot

after 2D gel electrophoresis with protein extract from

mock-trans-fected (1,2: 200, 400 lg) and p16INK4a-transfected cells (3,4: 200,

400 lg) Residual protein in gels was visualized by silver staining

(lower part of each panel) and each blotting procedure included a

positive control with Gal-3 (top right) (C) Quantitation of

cell-sur-face presentation of Gal-3 in mock-transfected (left) and p16 INK4a

-expressing Capan-1 pancreatic cancer cells (right) The control for

antigen-independent staining by omitting the incubation step with

the lectin-specific antibody from the protocol is given in each panel

(gray area), as are the percentage of positive cells and the mean

fluorescence intensity of staining when incubating with 20 lgÆmL)1

non-cross-reactive anti-galectin-3 Ig Standard deviation did not

exceed 11% in experimental series with four different experiments

run in triplicates.

when added to the medium As shown in Fig 3,

expo-sure of cells to Gal-3 was inhibitory in this

experimen-tal setting The percentage of p16INK4a-positive cells

undergoing anoikis after 20 h in suspension was

reduced when kept in the presence of suitable

concen-trations of Gal-3 To further strengthen the link

between Gal-3 and anoikis, the availability of this

lectin was upregulated deliberately by generating

p16INK4-positive cells, which additionally express Gal-3

at a high level via a second transfection Thus, the

presence of Gal-3 was increased, counteracting the

p16INK4a-dependent downregulation A series of five

clones was obtained with notably enhanced Gal-3

con-centration, an unspecific influence of the second

trans-fection step rendered unlikely by a mock control

(Fig 4A) The level of anoikis in these control cells

was used as a reference, and four Gal-3-overexpressing

clones revealed a trend towards reduced anoikis sus-ceptibility This reached statistical significance in clone Gal-3⁄ 2 (Fig 4B) Results showing that exogenous addition and vector-directed overexpression of Gal-3 can reduce the level of anoikis shaped the notion that Gal-3 physically hampers the induction of this cell death program In this case, even p16INK4a-negative cells should become sensitized toward anoikis if their Gal-3 production is downregulated This reasoning led

to the third set of experiments

To test the given hypothesis, we generated clones harboring an antisense vector for Gal-3 Because of this engineering, the clones contained a lower level of Gal-3 than wild-type and mock-transfected cell popula-tions (Fig 4C) Measurements of the corresponding cell-cycle profiles and percentages of cells undergoing anoikis revealed a Gal-3-dependent increase in this

0 200 400 0 200 400 600 0 200 400 600 0 200 400 600 FL2-H

5

A

B

C

FL2-H

Mock

p16-3

0

600

0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 600

Fig 2 The level of Gal-3 is diminished in p16 INK4a -expressing clones but remains constant over the 24 h anoikis induction period (A) Repre-sentative FACS histograms illustrating the increased extent of anoikis induction in Capan-1 ⁄ p16 INK4a -positive cells compared with a mock-transfected clone Cells were harvested following incubation on poly-HEMA to preclude attachment and trigger anoikis for the indicated time The given numbers indicate the percentage of cells with subdiploid DNA content (pre-G1 fraction) (B) Western blot analysis from extracts obtained under the conditions described above and analyzed with anti-Gal-3 or anti-PCNA Ig as indicated (C) Poly-(A + ) RNAs were isolated from mock-transfected and p16 INK4a -expressing clones and northern blots were probed for the presence of mRNA specific for Gal-3

or GAPDH (left) In vitro elongation of de novo RNA transcripts was performed in isolated nuclei from mock-transfected and p16INK4a -expressing clones in the presence of [ 32 P]UTP[aP], and the radioactively labeled RNA was hybridized to immobilized cDNA for Gal-3, GAPDH and b-actin (right) One representative of three experiments that yielded similar results is shown.

parameter (Fig 4D,E) Evidently, the level of Gal-3 is

important to protect wild-type cells from anoikis

induction, and the forced expression of Gal-3 in

p16INK4a-positive cells reduces their susceptibility to

anoikis

Because this pattern is inverse to the Gal-1-induced

effects reported previously [9], it is reasonable to

pro-pose functional competition between galectins-1 and -3

at the cell surface To probe for such an effect, we

stimulated anoikis by addition of Gal-1 to the culture

medium Should Gal-3 be an inhibitor, the extent of

Gal-1-dependent anoikis induction via glycan binding

will decrease Stepwise increases in the Gal-3

concen-tration progressively diminished the Gal-1-dependent

effect (Fig 5A), and galectin binding, shown to be

carbohydrate dependent [9], was reduced in

cross-com-petition assays (Fig 5B,C) The presence of Gal-3 can

thus impair the pro-anoikis effect of Gal-1 at the level

of the cell surface, here most likely targeting the

fibronectin receptor as had been shown in this and other carcinoma cell systems [9,27] If Gal-3 interferes with Gal-1 binding to and cross-linking the a5-subunit, then Gal-3 should also negatively affect post-binding signaling by the integrin We tested and established the involvement of caspase-8 activation for Gal-1-depen-dent anoikis induction (Fig 6A) and revealed a nega-tive impact of Gal-3 at this level (Fig 6B,C)

Discussion

The term ‘tumor suppressor’ summarizes an obvious function of a protein on malignancy Naturally, a sup-pressor engages secondary effectors at different levels, which turn its presence as a master organizer into ren-ormalization of phenotypic characteristics Because of growing insights into the role of glycosylation in cellu-lar communication, including growth control [6,28], we hypothesized that p16INK4ais capable of taking advan-tage of this network, explicitly by modulating the lectin reactivity of glycans and⁄ or lectin expression As

a consequence, further study provided a novel explana-tion for why this tumor suppressor restores susceptibil-ity to anoikis in Capan-1 pancreatic carcinoma cells [9] Having detected coordinated upregulation of both Gal-1 and suitable cell-surface ligands, the results provoked a question regarding orchestration of effects

on expression in the lectin family beyond Gal-1 Evi-dently, the chimera-type Gal-3, known as a functional antagonist of Gal-1 in a tumor model, offered a prime target for study

Cumulatively, the experiments reported herein revealed p16INK4a-dependent regulation of the presence

of Gal-3 in this cell system They also delineated a strong influence of Gal-3 on anoikis induction Of spe-cial note, we detected functional competition at the cell surface with the recently proven pro-anoikis activity of Gal-1, which involves caspase-8 activation Combining previous results on Gal-1⁄ a5b1-integrin co-immuno-precipitation, and the effects of p16INK4a on a5b1 -integrin, cell-surface glycosylation and Gal-1 [3,9] with the presented data enabled us to set up a scheme of p16INK4a-orchestrated changes that favor Gal-1-depen-dent anoikis (Fig 7) Whether and how a reduction in the intracellular activities of Gal-3 documented in other tumor cell types (e.g interaction with oncogenic K-ras and transcriptional modulation of cell-cycle reg-ulators such as cyclins A, D1and E as well as p21⁄ p27 [23,25,29]) will cooperate with the functional interfer-ence of cell-surface binding of homodimeric Gal-1, is not clear At the cell surface, the particular profile of the chimera-type galectin for ligand cross-linking shown recently [30] will definitely not elicit pro-anoikis

0

DNA

No Gal-3

+ 125 µg·mL –1 Gal-3

DNA

A

B

Fig 3 Addition of Gal-3 to the culture medium inhibits anoikis

induction (A) Gal-3 was added to p16 INK4a -expressing cells (clone

p16-3) in the concentrations given Following 20 h incubation on

poly-HEMA, anoikis rates were determined from DNA histograms

based on the fraction of cells with subdiploid DNA content The

data are expressed as percentage of the control without the

addi-tion of Gal-3 (n = 3, **P < 0.01, ***P < 0.005) (B) Representative

DNA histograms in the absence (no Gal-3) or in the presence of

125 lgÆmL)1Gal-3 The given numbers indicate the percentage of

cells in the pre-G1 fraction.

signaling of Gal-1 in this cell system This functional

competition between galectins, described initially for

SK-N-MC neuroblastoma cells and ganglioside GM1

[21], may well play a more general role in tumor

growth control Its detection gives a clear direction to

further research In this respect, such a study is

warranted on the p27-dependent downregulation of

carcinoma cell growth triggered by Gal-1 and most

likely the a5b1-integrin [27], as well as the Gal-1⁄

GM1-dependent communication between effector T and reg-ulatory T cells, which involves a4⁄ a5b1-integrins and

Ca2+ influx via TRPC5 channels [31] Eventually, these investigations will unravel the mostly unexplored intricacies of the galectin network monitored in tumors

by RT-PCR and by immunohistochemistry [32–34] Such cell biological studies will then shed light on additive⁄ synergistic, as opposed to antagonistic, activi-ties in malignancy and immune regulation

0

200 DNA

Wt

C

E

D

Fig 4 Upregulation of Gal-3 in clones changes anoikis susceptibility (A,B) Overexpression of Gal-3 protects p16INK4a-expressing clones from anoikis (A) Increased 3 content was confirmed via detection of 3 in western blots of clones that were stably transfected with a

Gal-3 expression construct Blots were conducted on whole-cell lysates of Capan-1 wild-type cells (wt), a p16 INK4a -expressing clone (p16) and p16INK4a-positive clones following mock transfection and with additional overexpression of Gal-3 (p16 ⁄ Mock and Gal-3 ⁄ 1-5) Blots were probed with anti-Gal-3 or anti-PCNA Ig (B) Determination of anoikis level in five p16 INK4a -positive clones with overexpression of Gal-3 and a respective mock control Extent of anoikis is expressed as a percentage relative to the mock-transfected control clone (n = 3, *P < 0.05) (C–E) Reduction in the level of endogenous Gal-3 stimulates anoikis induction (C) Downregulation of cellular Gal-3 was ascertained by detec-tion of Gal-3 in western blot analyses of protein extracts from wild-type (wt) and mock-transfected cells, as well as from two clones trans-fected with an antisense (as) Gal-3 cDNA construct (asG3A ⁄ B) Additional immunoblotting for PCNA (lower) was conducted to control for unspecific effects on protein synthesis (D) Determination of level of anoikis in clones with reduced Gal-3 Summary of anoikis rates obtained

in clones with decreased Gal-3 and mock controls Anoikis rates are given as a percentage of the total number of cells (n = 3, *P < 0.05,

**P < 0.01) (E) Representative cell-cycle histograms from cultures kept on poly-HEMA for 20 h The given numbers indicate the percentage

of cells in the pre-G1 fraction.

Equally important, our study broadens the basis for galectin involvement in tumor suppressor activity Here, the key reference point to date had been p53 In detail, SAGE screening on DLD-1 colon carcinoma cells expressing p53 identified galectin-7 as a p53-induced gene 1 in a group of 14 genes markedly upregulated from a total of 7202 tested transcripts [35] Along this line, genotoxic stress by UVB irradia-tion of human keratinocytes afforded a second system

in which a connection between p53 and galectin-7 was

200

A

B

C

**

**

*

*

#

##

150

50

Gal-3 binding (log fluorescence)

Gal-1 binding (log fluorescence)

10 0

10 0

Contr

ol

Gal-1 (100 µg·mL

–1 )

Gal-1 + Gal-3 (25 µg·mL

–1 )

Gal-1 + Gal-3 (50 µg·mL

–1 )

Gal-1 + Gal-3 (100 µg·mL

–1 )

Gal-1 + Gal-3 (150 µg·mL

–1 ) 0

100

Fig 5 Gal-3 inhibits Gal-1-stimulated anoikis and Gal-1 binding.

(A) Determination of Gal-1-stimulated (100 lgÆmL)1) anoikis in

p16INK4a-restituted cells in the presence of increasing

concentra-tions of Gal-3 Anoikis rates were determined following 20 h of

culture on poly-HEMA and were calculated based on the pre-G1

fraction from cell-cycle analyses Data are expressed as the

per-centage of vehicle-treated mock control cells (n = 4, *P < 0.05,

**P < 0.01, compared with control without Gal-1; #P < 0.05,

##P < 0.01 compared with data for Gal-1) (B) Binding of

biotiny-lated Gal-3 in the presence (light line) or absence (bold line) of

100 lgÆmL)1 Gal-1 The dashed line signifies the control with

fluorescent reagent, without prior incubation with biotinylated

Gal-3 (C) Binding of biotinylated Gal-1 in the presence (light line)

or absence (bold line) of 150 lgÆmL)1Gal-3 The dashed line

sig-nifies the control.

A Control 40

30 20 10

0

Time (h)

24

FAM-LETD-FMK

*

B

C

Fig 6 Gal-3 affects the onset of anoikis by inhibiting Gal-1-depen-dent caspase-8 activation (A) Anoikis after 6, 12 or 24 h incubation

on poly-HEMA in the presence or absence of the caspase 8 inhibi-tor FAM-LETD-FMK (B) Caspase 8 activation expressed as the per-centage of cells with active caspase 8 in the absence (control, filled circles) and presence (open circles) of 100 lgÆmL)1 Gal-3 (**P < 0.01, ***P < 0.001, compared with control) (C) Anoikis (percentage of cells in the pre-G1 fraction) in the absence (control, filled circles) and presence (open circles) of 100 lgÆmL)1 Gal-3 (**P < 0.01, compared with control).

made likely [36] When expressed in HeLa cell

transfec-tants, the intracellular function of galectin-7 appeared

to relate to affecting gene expression profiles

Conspic-uous changes were attributed to pro-apoptosis

signal-ing upstream of c-Jun N-terminal kinase activation

and cytochrome c release [37] When tested as an

extracellular effector, carbohydrate-dependent binding

led to growth inhibition of activated T cells or

neuro-blastoma cells via caspase-dependent or -independent

pathways [38,39] Of further relevance, the recently

documented effect of compensation of loss of

supres-sor genes in microsatellite instability on glycosylation,

including a2,6-sialylation of N-glycans, broadens the

scope for co-regulation [40] Further work to

strengthen this connection between a tumor suppressor

and lectin⁄ glycan remodeling as an effector pathway,

especially by examining clinical samples, is clearly

jus-tified

Examining galectin expression in clinical samples of

pancreatic cancer by gene-expression profiling

uncov-ered a consistent, albeit quantitatively variable,

upreg-ulation of Gal-3 expression [41–45] The similarly consistent upregulation of Gal-1 gene expression and protein production may at first seem puzzling Looking

at its immunohistochemical pattern, it could mostly be accounted for by a desmoplastic reaction around the tumor cells, with a Gal-1⁄ tissue plasminogen activator interaction being involved [44–50] Taking our data literally, an absence or low level of Gal-1, combined with an abundance of Gal-3 in the tumor cells, seems

to reflect impairment of p16INK4a As shown in the Supporting Information, preliminary immunohisto-chemical analysis on a limited number of cases focus-ing on the presence of p16INK4a and Gal-3 indicated a tendency for a negative correlation, in accordance with the in vitro data These results encourage thorough investigation of this aspect to strengthen clinical relevance By following this line of research, the orchestration of galectin expression described here may become instrumental in devising a novel therapeutic strategy to rationally shift the balance between resis-tance and susceptibility to favor the anoikis process

Materials and methods

Galectins

Human galectins were produced by recombinant expression, isolated by affinity chromatography on lactosylated Sepha-rose 4B as crucial step followed by gel filtration, and tested for purity using 1D and 2D gel electrophoresis and nano-electrospray ionization mass spectrometry, as well as for activity by hemagglutination [9,38,51,52] Biotinylation labeling was performed under activity-preserving conditions using the commercial N-hydroxysuccinimide ester derivative

of biotin (Sigma, Munich, Germany) Its incorporation into the galectins was determined using a proteomics protocol, and the activity of the labeled proteins was checked using carbohydrate-dependent solid-phase and cell binding assays [52,53] Polyclonal antibodies were raised in rabbits and rig-orously checked for lack of cross-reactivity using enzyme-linked immunosorbent assays and western blotting [54,55]

No experimental animals were used in this study Monitor-ing the cell-surface presentation of galectins was carried out

by flow cytofluorometry using fluorescent goat anti-rabbit IgG with 20 lgÆmL)1 galectin-type-specific IgG fractions and FACS equipment (Becton-Dickinson, Heidelberg, Germany) [9]

Cell culture

Human Capan-1 pancreatic carcinoma cells and clones with stable vector-directed p16INK4a expression were established and cultured as described previously [3]

Fig 7 Glycobiology of p16INK4afunctionality in Capan-1 pancreatic

carcinoma cells in vitro The tumor suppressor orchestrates: an

increase in the cell-surface presentation of the fibronectin receptor

(involving transcriptional upregulation of a 5 -subunit gene

expres-sion), regulation of glycogenes enabling increased Gal-1 cell-surface

reactivity, and an increase in the cell-surface presentation of Gal-1

(involving transcriptional upregulation of Gal-1 gene expression)

[3,9] Formation of Gal-1 ⁄ a 5 b 1 -integrin complexes with ensuing

cross-linking appears to lead to anoikis induction via caspase-8

activation Gal-3 can interfere with Gal-1 binding and⁄ or

Gal-1-dependent cross-linking at the level of the cell surface, decreasing

the pro-anoikis activity of Gal-1 p16 INK4a -dependent Gal-3

downre-gulation, with potential bearing also on intracellular anti-anoikis

activity of Gal-3, favors the pro-anoikis effect of cell surface Gal-1.

Antibodies to PCNA and p16INK4a were obtained from

Santa Cruz Biotechnology (Santa Cruz, CA, USA) and

NeoMarkers (Fremont, CA, USA), respectively

Protein extraction and western blotting

Cells were lyzed in radioimmunoprecipitation assay buffer

(50 mm Tris⁄ HCl at pH 7.5, 0.15 m NaCl, 0.25% SDS,

0.05% sodium deoxycholate, 1% NP-40, 1 mm

dithiothrei-tol, 1 lgÆmL)1 aprotinin, 2 mm leupeptin, 1 mm Na3VO4,

1 mm NaF, 1 mm phenylmethylsulfonyl fluoride), and

soni-cated on ice Aliquots (5–10 lg) were subjected to

SDS⁄ PAGE and electroblotted onto poly(vinylidene

fluo-ride) membranes (NEN, Cologne, Germany) Blots were

incubated overnight at 4C with the respective antibodies

(diluted 1 : 1000 in 5% non-fat dried milk in phosphate

buffered saline with 0.5% Tween-20) Immunoreactive

bands were visualized by enhanced chemoluminescence

(NEN) Cell processing, blotting and signal generation

when using 2D gel electrophoresis followed the procedure

previously used to detect Gal-1 [9]

Immunohistochemistry

Immunohistochemistry was performed on cryosections with

antibodies controlled for specificity and lack of intergalectin

cross-reactivity, as described previously [3,56,57] Briefly,

sections were fixed in 4% paraformaldehyde and primary

antibodies were applied at a dilution of 10 lgÆmL)1(Gal-3)

or 1.25 lgÆmL)1(p16INK4a) Immunoreactivity was detected

with biotinylated secondary antibodies and avidin–biotin–

peroxidase complex, with 3-amino-9-ethylcarbazole as the

chromogenic substrate Sections were counterstained with

hemalaun

Northern blot analysis

Total RNA from 108 cells was isolated using RNAzolB

(WAK-Chemie Medicals GmbH, Bad Homburg, Germany),

and the poly-(A+) fraction was purified using the

PolyA-Track System 1000 (Promega, Mannheim, Germany)

according to the manufacturer’s protocol Aliquots were

separated on a 1% agarose⁄

3-(N-morpholino)propanesulf-onic acid⁄ formaldehyde gel, blotted to Hybond N+

filters (Amersham Pharmacia, Freiburg, Germany) and linked to

the membrane using UV light Full-length cDNA

prepara-tions for human Gal-3 and glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) were labeled with [32P]dCTP[aP]

by random priming (Megaprime DNA labeling kit;

Amer-sham Pharmacia) Residual nucleotides were removed and

hybridization was carried out in Quick-Hyb buffer

(Strata-gene, La Jolla, CA, USA) at 65C for 2 h Following

hybridization, membranes were washed at 65C to a

strin-gency of 0.1· NaCl ⁄ Cit, 0.1% SDS and exposed to X-ray films at –70C

Nuclear run-off

Blots were prepared using isolated and denatured cDNA fragments immobilized on Hybond N+ nylon membranes (Amersham Pharmacia) The amount of denatured cDNA for Gal-1, GAPDH and b-actin blotted was 5, 2 and 1 lgÆslot)1, respectively Nuclear RNA preparation, labeling and hybrid-ization were performed as described previously [3] Blots were washed twice for 10 min at 42C with 40 mm NaH2PO4⁄

Na2HPO4 pH 7.2, 1% SDS Labeled de novo mRNA tran-scripts were detected on autoradiographs

Induction and detection of anoikis

For determination of anoikis, 2· 105

cells were cultured as suspension cultures in plates coated with poly(2-hydroxy-ethyl methacrylate) (poly-HEMA) (Sigma, Deisenhofen, Germany) for the indicated times Apoptotic cells were then quantitated from the pre-G1 fraction in cell cycle analyses

as described [3]

Stable transfection of Gal-3 sense/antisense cDNA

Full-length cDNA obtained from amplification of human Gal-3-specific mRNA of human DLD-1 colon carcinoma cells in either the sense (pcDNA–Gal-3S) or antisense (pcDNA–Gal-3AS) orientation was subcloned into the pcDNA3.1 vector, and the Effectene Transfection Reagent (Quiagen, Hilden, Germany) was used to generate stably transfected cells following the manufacturer’s protocol

Determination of binding of labeled galectins

Cells were incubated with 100 lgÆmL)1 of biotinylated Gal-1 and -3, washed and surface-bound probe was detected by flow cytometry using an indocarbocyanine– streptavidin conjugate Fluorescence intensity was recorded

on a FACSCalibur (Becton Dickinson) and analyzed with cellquest software [9,58] Cells incubated with the fluorescent indicator only were used to determine the back-ground fluorescence

Determination of caspase-8 activity

Cells with activated caspase-8 were detected using the carboxyfluorescelabeled derivative of the caspase-8

Hamburg, Germany), which irreversibly binds to activated caspase-8 Fluorescence intensity was evaluated by flow cytometry

Statistical analysis

Unless indicated, unpaired Student’s t-test analyses

(two-tailed distribution, two-sample unequal variance) were

per-formed using prism software (Prism, San Diego, CA,

USA) Data were considered significant at P-values < 0.05

Acknowledgements

This work is dedicated to Prof Dr Stefan Rosewicz

(1960–2004), who was crucial to start this project line

We are grateful to Drs B Friday, G Ippans and S

Namirha for helpful comments, to L Mantel for

excel-lent technical assistance as well as to the Dr Mildred

Scheel Stiftung, Sonnenfeld Stiftung, the

Wilhelm-San-der-Stiftung, LMUexcellent program, the Verein zur

Fo¨rderung des biologisch-technologischen Fortschritts

in der Medizin e.V and the EC program on Marie Curie

Research Training Networks (contract no

MRTN-CT-2005-019561) for generous financial support

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