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1
Comprehensive galectin fingerprinting in a
panel of 61 human tumor cell lines by RT-PCR
and its implications for diagnostic and thera-
peutic procedures
H
ARALD
L
AHM
1
, Sabine André
2
, Andreas Hoeflich
1
, Jürgen R. Fischer
3
, Bernard
Sordat
4
, Herbert Kaltner
2
, Eckhard Wolf
1
and Hans-Joachim Gabius
2
1
Institute of Molecular Animal Breeding, Gene Center, Feodor-Lynen-Strasse 25,
D-81377 Munich, Germany
2
Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Veterinär-
strasse 13, D-80539 Munich, Germany
3
Department of Medical Oncology, Thoraxklinik-Heidelberg gGmbH, Amalien-
strasse 5, D-69126 Heidelberg, Germany
4
Experimental Pathology Unit, Swiss Institute for Experimental Cancer Research
(ISREC), Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland
All correspondence and reprint requests should be addressed to:
Dr. Harald Lahm
Institute for Molecular Animal Breeding
Gene Center
Feodor-Lynen-Strasse 25
D-81377 Munich
GERMANY
Tel.: +49 - 89 - 2180 6815
FAX: +49 - 89 - 2180 6849
Email: lahm@lmb.uni-muenchen.de
2
Abstract
Purpose:
Knowledge about galectin expression by human tumor cells is mainly restricted to galec-
tins-1 and -3. This study was conducted to define the gene expression pattern of all presently known
human galectins in tumor cell lines of various histogenetic origin (galectinomics).
Methods:
The
presence of mRNAs for human galectins-1, -2, -3, -4, -7, -8 and -9 was monitored by RT-PCR anal-
yses in a panel of 61 human tumor cell lines of different origin (breast, colon, lung, brain, skin, kid-
ney, urogenital system, hematopoietic system).
Results:
The validity of the technique was first
confirmed by comparison of RT-PCR data with those obtained by Western blotting and cytofluo-
rometry for galectins-1 and -3 in 18 cell lines. The following detection of a complex pattern of gene
expression beyond commonly studied galectins-1 and -3 underscored the requirement for this fin-
gerprinting. The most abundantly expressed message for a member of this lectin family was galec-
tin-8 with 59 positive cell lines. With the exception of the tested lung tumors galectin-1 and -3
transcripts were frequently expressed in the cell line panel with differences between individual
cases. Positivity for galectins-2 and -4 was confined to a significant fraction of colorectal and neu-
ral tumors. Signals for galectin-9, the third known human tandem-repeat-type galectin besides -4
and -8, appeared in colorectal carcinoma cell lines with a frequency similar to that of galectin-4 but
with inter-line differences. Its expression was restricted to lines of this tumor type, of the tested
ovarian carcinoma and hematopoietic malignancies.
Conclusions:
The results clearly demonstrate
that human tumor cells express more mRNA species for galectins than those for galectins-1 and -3.
To derive unequivocal diagnostic and prognostic information by immunohistochemistry on galec-
tins with antagonistic impact on growth control and significant influence on cell adhesion addi-
tional monitoring of these so far understudied family members is essential.
Key words:
Apoptosis Galectin Human neoplasia Lectin Tumor
diagnosis Tumor growth control
3
Introduction
Elaboration of glycan antennae of glycoproteins and glycolipids is performed by a
complex enzymatic machinery. Sophisticated cloning strategies have provided
insights into an astounding diversity of the responsible gene families. β1,3(4)-
Galactosylation for example is performed by a total of at least 13 different cur-
rently known activities (Amado et al. 1999; Furukuwa and Sato 1999). The sheer
number of glycosyltransferases reflects a noticeable share of the coding genome
which is devoted to generation of these fine-structural details. Moreover, their reg-
ulation in differentiation argues strongly in favor of functions by their products in
biorecognition, a key reason to maintain the complexity of glycosylation (Reuter
and Gabius 1999). Concerning tumor biology, the profile of glycan structures is not
constant upon malignant transformation. In fact, tumor development and progres-
sion are frequently associated with aberrations of the sugar part of glycoconjugates
(Brockhausen et al. 1998; Hakomori 1998). Besides branching the ends of sugar
chains with derivative formation of the typical β-galactosides are often affected.
The encountered modifications can in the first step be considered as phenotypical
alterations of glycan biosynthesis. Since oligosaccharides surpass oligonucleotides
and -peptides by far in their capacity to store information (Laine 1997), these alter-
ations can also entail changes in the presentation of docking points for receptors
such as lectins (Gabius 2000; Rüdiger et al. 2000). If this reasoning has merit, it is
essential to prove the presence of endogenous lectins.
Lactose-inhibitable hemagglutination of mouse N-18 neuroblastoma cells was a
first decisive hint for the presence of lectin activity in mammalian tumor cells
(Teichberg et al. 1975). Biochemical purification proved their presence in tumors
and enabled to extend the analysis of their expression to Western blotting and
immunohistochemistry (Gabius et al. 1984, 1986a, b; Raz et al. 1984). The ensuing
investigations nourished the concept of lectins serving as functional tumor markers
(Gabius and Gabius 1990). From the five currently established categories of ani-
mal lectins (Gabius 1997a), the galectins deserve special attention in this respect.
They are specific for the mentioned spatially accessible glycan termini of cellular
glycoconjugates and exert a strong impact on cell growth and adhesion (Hiraba-
yashi 1997; Ohannesian and Lotan 1997; Zanetta 1997; Kaltner and Stierstorfer
1998; Perillo et al. 1998; André et al. 1999; Chiarotti et al. 1999; Cooper and Bar-
ondes 1999; Rabinovich 1999; Nangia-Makker et al. 2000). So far, studies with
tumors have centered on two rather prominent family members, i.e. the
homodimeric proto-type galectin-1 (with pro-apoptotic activity) and the chimera-
type galectin-3 (with anti-apoptotic activity). This work intimated prognostic rele-
vance of galectin expression in several cases but was also object of controversy
(for review, see Gabius 1997b; Itzkowitz 1997). One reason for concern is the
4
inherent restriction to only two, albeit often abundant, family members. The inter-
pretation of the results obtained for galectins-1 and -3 will only then be unequivo-
cal if no additional galectin(s) with overlapping or opposing functions is (are)
expressed in the tissue. Similar to the situation of diversification of glycosyltrans-
ferases cloning strategies were instrumental to define a series of human galectins
in addition to these two proteins. In detail, homodimeric proto-type galectin-2 and
also -7 (with low tendency for dimer formation) as well as the tandem-repeat-type
galectins-4, -8 and -9 are definitely expressed in human tissues (Hirabayashi et al.
1988; Cherayil et al. 1990; Gitt et al. 1992; Madsen et al. 1995; Su et al. 1996;
Hadari et al. 1997; Rechreche et al. 1997; Türeci et al. 1997; Matsumoto et al.
1998).
Several initial observations underscore that these galectins are not merely bystand-
ers in tumor biology but can bear upon our understanding of pathological pro-
cesses. Using the SAGE technique, the galectin-7 message belonged to 14 out of
7,202 transcripts in p53-expressing DLD-1 colorectal cancer cells before the onset
of apoptosis (Polyak et al. 1997), and the overexpression of this protein points to
its participation in UVB-induced apoptosis in epidermis (Bernerd et al. 1999). Dif-
ferential display of mRNA and cloning identified the rat homologue of human
galectin-7 as one of seven upregulated transcripts in chemically induced mammary
but not colonic carcinogenesis (Lu et al. 1997). Among the tandem-repeat-type
galectins, galectin-4 was detected as colon cancer antigen by an immunoscreening
of cDNA expression libraries (Scanlan et al. 1998), and galectin-8 effectively
interfered with integrin-mediated adhesion of non-small cell lung cancer cells of
the line 1299 (Hadari et al. 2000).
These findings emphasize that the presence of galectins beyond galectins-1 and -3
can influence the biological behavior of tumor cells. Consequently, the question
arises if and to what extent human tumor cell lines of different histogenetic origin
express these other members of the galectin family. By using the RT-PCR
approach we present clear evidence for a complex pattern of galectin expression in
different human tumors. The conclusion is drawn that further studies on galectins
in tumor diagnosis should be accompanied by such a fingerprinting to avoid over-
looking presence of a functionally important family member.
5
Materials and Methods
Cell lines and culture conditions
Table 1
The cell lines were cultured in RPMI 1640 supplemented with 10% FCS (all breast
and lung cancer cell lines, Colo201, Colo205, DLD-1, HCT-15, LNCaP, NIH-
OVCAR3, OAW-42, M07e, TF-1, THP-1), in DMEM supplemented with 5%
(Isreco-1) or 10% FCS (Isreco-2, -3, HCT-116, H4, Hs683, SW1088, SW1783,
U118, Hs294T, ACHN, SW13, 293), in EMEM supplemented with 10% FCS
(T98G, U87, U373, DU145) or in a 1:1 mixture of DMEM/Ham’s F-12 (PC-3 and
remaining colorectal carcinoma cell lines). All culture media contained L-
glutamine (final concentration of 2 mM). The factor-dependent cell lines M07e
and TF-1 were cultured in the presence of 10 ng/ml granulocyte-macrophage col-
ony-stimulating factor. For T-47D and NIH-OVCAR3 10 µg/ml insulin were
added to the culture medium. The normal colonic cell line was cultured in a special
DMEM/Hepes/glutamine medium (GIBCO, Karlsruhe, Germany, Cat. No. 12340-
021) supplemented with 10% FCS, 1 nM epidermal growth factor, amino acids,
vitamins and antibiotics as described (Deveney et al. 1996). The HPR600 and
Lisp-1 cell lines were provided by Drs. H.-P. Rutz (CHUV, Lausanne, Switzerland)
and D. Lopez (São Paulo, Brazil). The normal human colonic epithelial cell line
was received from Dr. U. Wenzel (Weihenstephan, Germany). The small cell lung
cancer (SCLC) cell lines were provided by Dr. G. Bepler (Duke University Medi-
cal Center, Durham, NC). The HaCaT cell line (Boukamp et al. 1988) was a gift of
Drs. A. Villalobo and M. Quintanilla (Instituto de Investigaciones Biomédicas,
Madrid, Spain). The other cell lines had been established at ISREC, Epalinges,
Switzerland or were obtained either from ATCC (Manassas, VA), DSMZ (Braun-
schweig, Germany) or the tumor collection of the DKFZ (Heidelberg, Germany).
All cell lines used in this study are listed in Table 1.
Cytofluorometric measurements
For the detection of cell surface expression of galectins non-crossreactive poly-
clonal anti-galectin-1, -3 and -8 antibodies were used in a staining protocol
described in detail previously (André et al. 1999). Briefly, tumor cells were thor-
oughly washed with Dulbecco’s phosphate-buffered saline solution containing
0.1% bovine serum albumin to block any non-specific protein-binding sites prior
to the incubation of cell-containing aliquots (4×10
5
cells in 50 µl) with antibody
fraction (20 µg/ml) for 30 min at 4°C. Washing, incubation with the indicator com-
plex FITC-conjugated goat anti-rabbit IgG (Sigma, Munich, Germany; 1:100 dilu-
tion) and further washing to remove unbound indicator were followed by fixation
6
with 1% paraformaldehyde overnight and the quantitative assessment in a FACS-
can instrument (Becton-Dickinson, Heidelberg, Germany) equipped with the soft-
ware CONSORT 30. Intracellular galectin presence was determined by confocal
laser scanning microscopy (equipment type LSM510 from Zeiss, Jena, Germany)
and pre-staining cell fixation in the presence of 0.1% Triton X-100 again using the
fluorescent second antibody as indicator substance.
RNA extraction and RT-PCR analysis
Cells were grown to subconfluence in petri dishes and were lysed by the addition
of Tri-Pure™ isolation reagent (Roche Diagnostics, Mannheim, Germany). Tumor
tissue and normal colon tissue of patients HO672 and 899 were stored at -80°C
after surgery in the tumor bank of the Klinikum Grosshadern (Germany). The sam-
ples were kindly provided by Dr. M. Heiss (Klinikum Grosshadern, Germany).
Frozen pieces were put in Tri-Pure™ isolation reagent and minced in a homoge-
nizer. Total RNA was prepared according to the manufacturer’s recommendation.
Prior to cDNA production total RNA preparations were incubated for 30 min at
37°C with RNase-free DNase I to digest residual genomic DNA (Roche Diagnos-
tics). The reaction was stopped by incubation for 10 min at 75°C. As a template for
cDNA synthesis 2.5 µg total RNA were used. Reverse transcription was performed
for 60 min at 37°C in RT buffer (50 mM Tris/HCl, pH 8.3, 75 mM KCl, 3 mM
MgCl
2
), 10 mM DTT, dNTPs (1 mM each), random hexamer primers (0.6 µg) and
20 U M-MuLV reverse transcriptase (GIBCO). The reaction was terminated by
incubation for 10 min at 95°C.
Table 2
Subsequent PCR analyses were carried out in 20 µl reactions containing 2 µl
cDNA, 0.5 U
Taq
polymerase (Peqlab Biotechnologie GmbH, Erlangen, Ger-
many), 50 mM KCl, 10 mM Tris/HCl (pH 8.8), 1.5 mM MgCl
2
, dNTPs (50 µM
each) and 0.1 µM of both sense and anti-sense primers. For detection of galectin-7
transcripts using the HaCaT keratinocyte line as a positive control a
Taq
poly-
merase from Qiagen (Hilden, Germany) proved to be most appropriate. The reac-
tion conditions were almost identical except that a Q solution (provided by the
supplier) and 3 mM MgCl
2
had to be used. The sequence and location of the prim-
ers used and the calculated lengths of the products are depicted in Table 2. To min-
imize the risk of amplification of residual genomic DNA sequences the sequence
stretch between primer positions was deliberately chosen to be very large to
include - if possible - a sizeable fraction of the introns. Amplification of all galec-
tin-specific transcripts was run as follows: samples were heated to 94°C for 4 min
followed by 36 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 2 min. After
a final extension period of 10 min at 72°C amplified products were separated in
2% TAE gels and visualized by ethidium bromide staining under UV light.
7
Results
Comparison between results of RT-PCR analysis and detec-
tion of the translated protein
Figure 1
Table 3
RT-PCR analysis is highly sensitive and entails visualization of even scarcely
abundant messages. As internal calibration to what extent positive signals by this
method are indicative of actual presence of the respective galectin in human tumor
cells we first selected cell lines for which galectin expression on the level of pro-
tein expression had been demonstrated. As compiled in Table 3, Western blotting
and/or cytofluorometry had been carried out to detect galectins-1 and -3 in extracts
or on surfaces of human tumor cells growing in suspension or adherent to tissue
culture flasks (Nangia-Makker et al., 1995; Ohannesian et al., 1995; André et al.,
1999; Ellerhorst et al., 1999a). The extent of cell surface presentation of these
galectins and also of galectin-8 is exemplarily illustrated for Colo201 cells (Fig. 1).
Further confocal laser scanning microscopy revealed additional cytoplasmic stain-
ing for galectins-1 and -3, the latter also present in cell nuclei (not shown). Evi-
dently, this compilation served as a crucial test for the predictive accuracy of the
RT-PCR analysis.
Figure 2
To exclude any interference of genomic DNA as template with ensuing false-posi-
tive signals we first performed assays to examine the success of primer positioning
to prevent amplification of genomic DNA sequences. Indeed, no signal was
obtained except for galectin-7. In this case, an amplification product of approxi-
mately 1.3 kb was produced, which was consistently absent after the routine
DNase I treatment. By the way, its sequencing confirmed the presence of an intron
of about 800 bp between the coding sequence (not shown). Using the primer sets
compiled in Table 2, we strived for restricting amplification deliberately to the
selected galectin. As a further control of specificity, the length of each product was
calculated according to the published cDNA sequence (Table 2), and any deviation
from this number will be reason for concern. As shown in Fig. 2 for selected cell
lines the amplification of individual galectin-specific transcripts was selective. In
the cases of galectins-1 and -3 the anticipated fragment lengths of 321 bp and 719
bp, respectively, were in fact ascertained. Having established this experimental
basis, we performed monitoring of expression of mRNAs for galectin-1 and -3
with the 18 cell lines with known protein expression profiles. Without exception
the RT-PCR data coincided with previous experiments measuring the level of
expression of these two proteins (Table 3). This match between the different meth-
8
ods attests the reliability and prompts to proceed with the intended fingerprinting.
Nevertheless, a note of caution is warranted, comprising that this perfect agree-
ment should not be misinterpreted to guarantee presence of the respective galectin
at an appreciable amount in every single case presented in the following para-
graphs.
Detection of transcripts for the panel of human galectins in
tumor cells
Having first ascertained the technical reliability of RT-PCR analysis and the corre-
spondence to protein expression in the cases of galectins-1 and -3, the two most
commonly studied human galectins in cancer research, we next extended our mon-
itoring to the complete set of currently known human galectins. The distinctive
primer sets and all calculated fragment sizes are compiled in Table 2. As represen-
tatively shown in Fig. 2, our assumption that galectin expression in human tumors
extends beyond galectin-1 and -3 was experimentally confirmed. Transcripts for
other galectins with exactly the calculated fragment lengths were visible in the gels
(Fig. 2). Interestingly, one deviation from the calculations based on published
cDNA sequences was put on record. Similar to the case of galectin-9 investigated
in detail in our previous report (Lahm et al. 2000), an isoform with a sequence
addition was observed for galectin-8 (Fig. 2). Its exact molecular nature is cur-
rently being studied. The overall non-uniform pattern of transcripts shown in this
figure prompted the profiling in an array of established human tumor cell lines of
different histogenetic origin (see Table 1 for compilation of selected lines).
Profile of galectin-specific transcripts in breast cancer cells
Table 4
Previous immunohistochemical analysis for galectins-1 and -3 had supported the
prediction of gene transcription for at least these two galectins in breast cancer
(Gabius et al. 1986a). Remarkably, transcript presence within the galectin family
was not confined to these two family members. Galectin-8 gene expression could
be defined as a further consistent feature (Table 4). Also, the transcript for galectin-
7 was present in the HuMI line and its tumorigenic sublines (Yilmaz et al. 1993)
and DU4475 cells. Neither transcripts coding for galectin-2 nor the two forms of
galectin-9 were detected (Table 4). Likewise, no indication for transcription of the
galectin-4 gene was obtained.
Profile of galectin-specific transcripts in colon cancer cells
Since galectin-4 is present in the murine and human gastrointestinal tract, we antic-
ipated to see its presence in colon cancer lines, along with galectins-1 and -3, as
9
already documented in Table 3. Indeed, 14 of 22 cell lines displayed an RT-PCR
signal specific for the galectin-4 transcript (Table 4). Amplification also yielded
this signal in the normal colonic cell line. Among the tandem-repeat galectins the
appearance of positivity for the two galectin-9-specific transcripts was a further
distinguishing factor from breast carcinomas. Galectin-2 or -7 transcripts were
found in several instances in galectin-1-negative cell lines. Positivity for galectin-3
and -8 transcripts appeared as a common feature, as noted for the breast cancer
lines (Table 4). The conclusion on this being a general property of malignant cells,
however, is not justified as shown by lung tumor lines.
Figure 3
To indicate whether the results on cell lines have relevance for the clinical situa-
tion, we included specimen from two colorectal cancer patients into our analysis.
We have compared galectin gene expression in samples of neoplastic and the cor-
responding normal colonic tissue, one representative result illustrated in Fig. 3.
Although the number of cases is too limited for far-reaching conclusions, the gene
expression profiles of the tumors can be reconciled with those of colon cancer cell
lines. Interestingly, transcripts coding for insertional isoforms of galectins-8 and -9
were detected in both samples, revealing that these isoforms are also expressed
in
vivo
and do not merely represent an attribute of cultured cell lines.
Profile of galectin-specific transcripts in lung carcinoma
cells
While the galectin-8-specific transcripts was frequently encountered, positivity for
galectin-3 was only assessed in 5 of 10 lines (Table 4). Similarly, the galectin-1
transcript was present in 4 lines, and a signal for the galectin-7 transcript was
delineated in only two cases of small cell lung cancer cell lines. The absence of
transcripts specific for example for galectin-4 or -9 might be exploitable in diagno-
sis to infer origin of metastatic lesions. To what extent the gene activity in neural
tumors of the selected panel differs from the studied carcinomas is presented in the
next paragraph.
Profile of galectin-specific transcripts in brain tumor cells
Cell lines derived from malignancies of the central nervous system consistently
and strongly expressed galectin-1-, -3- and -8-specific transcripts (Table 4). Addi-
tionally, at least one line was positive for the galectin-2 gene transcript, and it is
noteworthy that mRNA preparations from three lines yielded the galectin-4-spe-
cific signal, although signals in two cases were weak and not consistently detect-
able. No evidence for the presence of mRNA for galectin-7 could be gathered,
whereas the amplification of galectin-9-specific transcripts reached a level and fre-
10
quency to justify to include (+) into Table 4.
Profile of galectin-specific transcripts in tumor cells of mis-
cellaneous origin
To furnish information on additional tumor cell types, we added a collection of 12
further lines of different histogenetic origin to our study. This series of measure-
ments substantiated that galectin-8 gene expression is a prominent characteristic of
human tumor cells (Table 4). The detection of the prostate carcinoma tumor anti-
gen PCTA-1 with 97% homology on the protein level to galectin-8 by immunoflu-
orescence and immunoprecipitation analysis of the three prostate cancer lines (Su
et al. 1996) served as further inherent control for the validity of the RT-PCR data
(Table 3). Moreover, the three data sets from prostate carcinoma cells underscored
the potential for non-uniform expression. Remarkably, the cells of the LNCaP line,
which is poorly adhesive in culture and non-invasive
in vitro
, was negative for
galectin-1 and -3. The ovarian carcinoma and the hematopoietic tumor cell lines
shared galectin-9 but not galectin-4 (except for THP-1 cells) gene expression with
colon cancer lines (Table 4).
11
Discussion
In the present study we have investigated galectin gene expression of all presently
known human members of this lectin family in neoplastic cells. For the first time
the complete galectin expression pattern was determind in a panel of 61 tumor cell
lines of different histogenetic origin. To date studies in this area have nearly exclu-
sively focused on galectins-1 and -3. Our results clearly show that this restriction
can confound the interpretation. In addition, they point toward the possibility of a
tissue-related profile of certain galectins useful e.g. to delineate the origin of meta-
static lesions. For diagnostic approaches this detailed fingerprinting is the refine-
ment of initial glycohistochemical detection of a galectin activity by
neoglycoconjugates with lactose or N-acetyllactosamine as pan-galectin ligand
(Gabius 1989). Furthermore, the determination of such individual patterns pro-
vides the basis for the design of forthcoming transfection experiments with sense
and anti-sense expression vectors. The resulting cell systems will contribute to elu-
cidate the biological function of distinct galectins. In fact, the extent of overlap-
ping and antagonistic activities on growth control and cell adhesion of the products
of gene diversification in the galectin family is still largely unknown.
Diversification within the family of mammalian galectins has given rise to a group
of proteins with a homologous carbohydrate recognition domain also in human tis-
sues (Cooper and Barondes 1999). Interestingly, their cellular localization can dif-
fer implying different targeting mechanisms, as shown for T84 colon
adenocarcinoma cells with galectins-3 and -4 residing at the apical or basal mem-
brane, respectively (Danielsen and van Deurs 1997; Huflejt et al. 1997). In addi-
tion to differences in the distribution in subcellular compartments two main
characteristics strongly argue in favor of distinct functions for individual galectins.
First,
in vivo
ligand selection between galectins has non-uniform properties,
although the already carefully mapped ligand profiles of galectins-1 and -3 overlap
to a notable degree (Sparrow et al. 1987; André et al. 1999; Hadari et al. 2000;
Matsushita et al. 2000). Engineered ligand derivatives are instrumental to delineate
such alterations in the architecture of the combining sites (Solís et al. 1996; Rüdi-
ger et al. 2000). Histochemically, these differences are started to be exploited in
tumor diagnosis by the introduction of labeled galectins as tools in pathology
(André et al. 1999; François et al. 1999; Schwarz et al. 1999; Delorge et al. 2000;
Plzák et al. 2000).
The second distinctive feature concerns spatial orientation of the binding sites due
to its impact on triggering biosignaling by crosslinking carbohydrate ligands (Vil-
lalobo and Gabius 1998). Presentation of the two binding sites at opposing sections
of the human lectin is shared by the homodimeric (proto-type) galectins-1, -2 and -
7 (the latter one with tendency to form monomers) and the tandem-repeat-type
12
galectins-4, -8 and -9 (Gabius 1997a; Hirabayashi 1997). Galectin-3 is unique in
this respect owing to its chimeric nature by joining a collagenase-sensitive stalk to
the typical carbohydrate recognition domain (Hughes 1993). These differences in
ligand selection and/or crosslinking and also the varying degrees to be engaged in
additional protein-protein interactions explain why two galectins (i.e. galectins-1
and -3) even elicit antagonistic responses on a cellular parameter, i.e. induction of
or resistance to apoptosis (Yang et al. 1996; Akahani et al. 1997; Perillo et al. 1998;
Kim et al. 1999; Rabinovich 1999).
As the examples of galectins-7 and -8 underscore, which have already been men-
tioned in the introduction (Polyak et al. 1997; Bernerd et al. 1999; Hadari et al.
2000), these family members also harbor potential to participate in growth regula-
tion. Although it is unclear yet whether this activity plays any role for the prognos-
tic relevance of binding of histo-blood group A-trisaccharide-exposing
neoglyoconjugates (
in vitro
binding partner of galectins) in primary non-small cell
lung cancer and metastases to the lung (Kayser et al. 1994, 1998), it is evident at
this stage that any implication on a role of galectins for the course of the disease
should be based upon the complete panel of galectin presence. By all appearances,
this notion is strongly supported by the remarkable functional role of an individual
galectin in stable cell lines transfected with an expression vector for either galec-
tin-1 or -3 (Ellerhorst et al. 1999a; Matarrese et al. 2000a, b).
One instructive model in this respect is furnished by the prostate cancer line
LNCaP, which is negative for expression of galectins-1 and -3 (Tables 3 and 4).
Overexpression of galectin-1 triggers notable effects, i.e. acceleration of adhesion
to the extracellular matrix glycoproteins laminin and fibronectin, two proven
galectin ligands, growth inhibition and induction of apoptosis (Ellerhorst et al.
1999a, b). Similarly, stable galectin-3 transfectants of the BT-549 breast carcinoma
cell line adhered much more rapidly to laminin and collagen IV than the tumor
cells devoid of this galectin (Warfield et al. 1997). A three-fold enhancement in
matrigel invasion of the transfectant corresponds with observations on breast carci-
noma cell lines from various stages of disease progression that exogenous supply
of galectin-3 could stimulate migration through an extracellular matrix for early-
disease-stage cells (Le Marer and Hughes 1996; Warfield et al. 1997). The fact that
the surface presentation of α
6
β
1
- or α
4
- and β
7
-integrins was elevated in galectin-3
transfectants may contribute to account for the pronounced effect on adhesive
properties (Warfield et al. 1997; Kim et al. 1999; Matarrese et al. 2000a). For
LSLiM6 and HM7 colon cancer cells, galectin-3 up- and downregulation corre-
lated positively with the metastatic capacity of the cells to the liver (Bresalier et al.
1998).
BT-549 transfectants acquired anchorage-independent growth properties after sta-
ble integration and expression of the galectin-3 cDNA in formerly galectin-3-nega-
13
tive cells (Nangia-Makker et al. 1995). Intracellularly, galectin-3 appeared to
protect the cells against damage and death induced by alteration of the mitochon-
drial membrane potential and reactive oxygen species (Matarrese et al. 2000b). In
aggregate, these studies demonstrate that modulation of galectin expression is an
event readily visible on the level of cell growth and invasiveness. To contribute to
predict cell features on this basis and then to exploit especially the capacity of
galectins for mediation of negative growth regulation critically depends on the
knowledge of presence of the family members.
The salient lesson that emerges from this study is the ability of tumor cells to
express transcripts for the known human galectins without a restriction to galec-
tins-1 and -3. Consequently, immunohistochemical monitoring of galectins will
have to be extended to prevent misinterpretation. Moreover, the presented results
provide a guideline to devise transfection schemes to elucidate and, if promising,
to exploit functional aspects of individual lectins. Stable transfectants with sense
and anti-sense constructs will have to be examined to complement histopathologi-
cal work. Results of these studies are essential for the conclusion on how individ-
ual galectins in tumor cells cooperate or are assigned to different functions. Work
to establish the required expression constructs and to determine cellular respon-
siveness to exogenous supply of galectins is in progress.
Acknowledgements
We are indebted to Dr. A. Villalobo and Dr. M. Quintanilla (Madrid, Spain) for
kindly providing the HaCaT cell line as a positive control for the establishment of
the galectin-7 PCR. We thank Dr. M. Heiss (Munich, Germany) for providing us
with patient samples of normal and neoplastic colonic tissue. We also gratefully
acknowledge the excellent technical assistance of A. Helfrich and the generous
financial support of the Wilhelm Sander-Stiftung.
14
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19
Figures
Fig. 1 Detection of cell-membrane-associated galectins.
Flow cytometry histograms of cell surface staining of Colo201 colon adenocarcinoma cells using
anti-galectin-1 (A), anti-galectin-3 (B) and anti-galectin-8 (C) immunoglobulin G fractions. Dotted
lines represent the control without the incubation step with the primary antibody, as described in
Materials and Methods.
20
Fig. 2 Detection of galectin-specific transcripts in different human tumor cell lines by RT-PCR
analysis.
cDNA preparations of cell lines were subjected to amplification by galectin-specific primers. Num-
bers on the left indicate the length of molecular weight markers (M). aq. bidest.: negative control in
which water was used as a template.
HuMI
hColon
T98G
THP-1
Gal-1
Gal-2
Gal-3
Gal-4
Gal-7
Gal-8
Gal-9
aq. bidest.
SCLC-22H
Colo201
883
500
242
883
500
242
883
500
242
883
500
242
883
500
242
883
500
242
M
HuMI
hColon
T98G
THP-1
Gal-1
Gal-2
Gal-3
Gal-4
Gal-7
Gal-8
Gal-9
aq. bidest.
SCLC-22H
Colo201
883
500
242
883
500
242
883
500
242
883
500
242
883
500
242
883
500
242
HuMI
hColon
T98G
THP-1
Gal-1
Gal-2
Gal-3
Gal-4
Gal-7
Gal-8
Gal-9
aq. bidest.
Gal-1
Gal-2
Gal-3
Gal-4
Gal-7
Gal-8
Gal-9
aq. bidest.
SCLC-22H
Colo201
883
500
242
883883
500500
242242
883
500
242
883883
500500
242242
883
500
242
883883
500500
242242
883
500
242
883883
500500
242242
883
500
242
883883
500500
242242
883
500
242
883883
500500
242242
M
21
Fig. 3 Galectin gene expression in normal and malignant human colon cells.
cDNA of normal colon and tumor tissue from patient HO672 were subjected to galectin-specific
RT-PCR analyses. Numbers on the left indicate the length of molecular weight markers (M). aq.
bidest.: negative control in which water was used as a template.
883
500
242
normal
883
500
242
tumor
Gal-1
Gal-2
Gal-3
Gal-4
Gal-7
Gal-8
Gal-9
aq. bidest.
M
883
500
242
normal
883
500
242
tumor
883
500
242
normal
883
500
242
883883
500500
242242
normal
883
500
242
tumor
883
500
242
883883
500500
242242
tumor
Gal-1
Gal-2
Gal-3
Gal-4
Gal-7
Gal-8
Gal-9
aq. bidest.
M
Gal-1
Gal-2
Gal-3
Gal-4
Gal-7
Gal-8
Gal-9
aq. bidest.
M
22
Table 1: List of investigated tumor cell lines
Cell line
Origin
a
Breast cancer
HuMI
*b
immortalized mammary epithelial
cells
HuMI-T
*b
weakly tumorigenic HuMI cells
TTu1
*b
highly tumorigenic HuMI subline
TTu2
*b
highly tumorigenic HuMI subline
BT-20 HTB19 mammary gland adenocarcinoma
DU4475 HTB123 mammary gland adenocarcinoma
MDA-MB-468 HTB132 adenocarcinoma
T-47D HTB133 ductal carcinoma
ZR-75-30 CRL1504 ductal carcinoma
Colorectal carcinoma
Caco-2 HTB37 adenocarcinoma
Co115
c
carcinoma
Colo201
*
CCL224 adenocarcinoma (ascites)
Colo205
*
CCL222 adenocarcinoma (ascites)
DLD-1 ACC278† adenocarcinoma
HCT-15 ACC357† adenocarcinoma
HCT-116 CCL247 carcinoma
HPR600
d
carcinoma
HT29 HTB38 adenocarcinoma
Isreco-1
*e
primary tumor
Isreco-2
*e
liver metastasis
Isreco-3
*e
peritoneal metastasis
Lisp-1
d
carcinoma
LoVo CCL229 supraclavicular metastasis
LS174T CL188 adenocarcinoma
LS411N CRL2159 carcinoma
23
LS513 CRL2134 carcinoma
LS1034 CRL2158 carcinoma
SW480
*
CCL228 adenocarcinoma
SW620
*
CCL227 lymph node metastasis
SW1116 CCL233 adenocarcinoma
WiDr CCL218 adenocarcinoma
hColon
f
normal colonic cell line
Table 1: List of investi-
gated tumor cell lines
(cont'd)
Lung carcinomas
NCI-H69 HTB119 SCLC, carcinoma
NCI-N417 CRL5809 SCLC, carcinoma
NCI-N592 CRL5832 SCLC, carcinoma (bone marrow
metastasis)
SW210.5
g
SCLC, carcinoma
SCLC-16HV
g
SCLC, carcinoma
SCLC-21H
*
ACC372† SCLC, carcinoma
SCLC-22H
*
ACC373† SCLC, carcinoma
SCLC-24H
h
SCLC, carcinoma
HS-24 ‡ NSCLC, squamous cell carcinoma
SB-3 ‡ NSCLC, suprarenal gland metastasis
Neural tumors
H4 HTB148 neuroglioma
Hs683 HTB138 glioma
SW1088 HTB12 astrocytoma
SW1783 HTB13 astrocytoma
T98G CRL1690 glioblastoma
U87 HTB14 glioblastoma/astrocytoma
U118 HTB15 glioblastoma/astrocytoma
Table 1: List of investigated tumor cell lines
Cell line
Origin
a
24
a
If not otherwise stated the numbers refer to the code of the American Type Cul-
ture Collection (Manassas, VA)
† DSMZ number (German Collection of Microorganisms and Cell Cultures,
Braunschweig)
‡ Tumor cell collection of the DKFZ (German Cancer Research Center, Heidel-
berg)
*
These cell lines were derived from a single patient
b
Yilmaz et al. (1993),
c
Carrel et al. (1976),
d
for the origin of these cell lines see
Materials and Methods,
e
Cajot et al. (1997),
f
Deveney et al. (1996),
g
Bepler et al.
(1988),
h
Bepler et al. (1989)
U373 HTB17 glioblastoma/astrocytoma
Miscellaneous tumor
types
Hs294T HTB140 melanoma
ACHN CRL1611 renal cell adenocarcinoma
SW13 CCL105 adrenal gland carcinoma
DU145 HTB81 prostate carcinoma
LNCaP CRL1740 prostate carcinoma (supraclavicular
lymph
node metastasis)
PC-3 CRL1435 prostate adenocarcinoma (bone
metastasis)
NIH-OVCAR3 HTB161 ovarian adenocarcinoma
OAW-42 ‡ ovarian carcinoma
M07e ACC104† acute megakaryoblastic leukemia
TF-1 CRL2003 erythroleukemia
THP-1 TIB202 acute monocytic leukemia
293 CRL1573 transformed embryonic kidney fibro-
blasts
Table 1: List of investigated tumor cell lines
Cell line
Origin
a
25
Table 2: Primer sequences used in RT-PCR analyses
Primer Sequence
Location
a
Size
b
Galectin-1#1 AAC CTG GAG
AGT GCC TTC
GA
45 – 64 321 bp
Galectin-1#2 GTA GTT GAT
GGC CTC CAG
GT
367 – 348
Galectin-2#1 ATG ACG GGG
GAA CTT GAG
GTT
27 – 47 358 bp
Galectin-2#2 TTA CGC TCA
GGT AGC TCA
GGT
384 – 364
Galectin-3#1 ATG GCA GAC
AAT TTT TCG
CTC C
1 – 22 719 bp
Galectin-3#2 ATG TCA CCA
GAA ATT CCC
AGT T
719 – 698
Galectin-4#1 GCT CAA CGT
GGG AAT GTC
TGT
101 – 121 609 bp
Galectin-4#2 GAG CCC ACC
TTG AAG TTG
ATA
709 – 689
Galectin-7#1 ATG TCC AAC
GTC CCC CAC
AAG
19 – 39 282 bp
Galectin-7#2 TGA CGC GAT
GAT GAG CAC
CTC
300 – 280
Galectin-8#1 GTT GTC CTT
AAA CAA CCT
ACA G
45 – 66 608 bp
Galectin-8#2 TAA CGA CGA
CAG TTC GTC
CAG
652 – 632
26
Galectin-9#1 ACT ATT CAA
GGA GGT CTC
CAG
145 – 165 571 bp
Galectin-9#2 GGA TGG ACT
TGG ATG GGT
ACA
715 – 695
a
Numbers desi-
gnate the base
pair positions
according to
published cDNA
sequences
b
Predicted size of
the correct frag-
ment
Table 2: Primer sequences used in RT-PCR analyses
Primer Sequence
Location
a
Size
b
27
a
determined for colon cancer lines by Ohannesian et al. (1995), for the breast
cancer line by Nangia-Makker et al. (1995) and for the three prostate cancer
lines by Ellerhorst et al. (1999a)
b
determined by André et al. (1999) (except for Colo201, this study, and the
three prostate cancer lines, presented by Ellerhorst et al. (1999a))
ND: not determined
Table 3: Detection of expression of galectins-1 and -3 by different methods
Cell line
Western
blot
a
FACS
Analysis
b
RT-PCR
Gal-1 Gal-3 Gal-1 Gal-3 Gal-1 Gal-3
Caco-2 – + ND ND – +
Colo201 ND ND + + + +
Colo205 ND ND + + + +
DLD-1 – + ND ND – +
HCT-15 – + ND ND – +
HCT-
116
+ + ND ND + +
HT29 – + ND ND – +
LoVo – + ND ND – +
LS174T – + ND ND – +
SW480++++++
SW620++++++
T-47D++++++
HS-24NDND++++
SB-3NDND++++
DU145 + + + – + +
LNCaP––––––
PC-3+++–++
NIH-
OVCAR
3
ND ND (+) (+) + +
28
Table 4: Galectin gene expression by human tumor cell lines
Gal-1 Gal-2 Gal-3 Gal-4 Gal-7 Gal-8
Gal-9
a
(A)
Gal-9
a
(B)
Breast tumors
HuMI +–+–++– –
HuMI-T +–+–++– –
TTu1 +–+–++– –
TTu2 +–+–++– –
BT-20 +–+––+– –
DU4475 + – + – + + – –
MDA-MB-468+–+––+– –
T47D +–+––+– –
ZR-75-30 + – + – – + – –
Colon tumors
Caco-2 –++–++– –
Co115 ––+–+++ +
Colo201 + – + + + + + –
Colo205 + – + + + + + –
DLD-1 – – + + – + – –
HCT-15 ––+–++– –
HCT-116+–+–++– –
HPR600 –+++–++ +
HT29 –++++++ +
Isreco-1 + – + – + + + +
Isreco-2 + (+) + + + + + +
Isreco-3 – – + + + + + +
Lisp-1 +–+–+++ +
LoVo ––++++– –
LS174T – – + + – + + –
LS411N – + + + – + + +
LS513 ––++–++ +
29
LS1034 – + + + – + + +
SW480 +–+–++– +
SW620 +–+–++– +
SW1116 – – + + + + – –
WiDr –++++++ +
hColon + + + + + + – –
Lung tumors
NCI-H69–––––+– –
NCI-N417 + – (+) – – – – –
NCI-N592–––––+– –
SW210.5 – – – – + + – –
SCLC-16HV––––++– –
SCLC-21H––+––+– –
SCLC-22H+–+––+– –
SCLC-24H––––––– –
HS-24 +–+––+– –
SB-3 +–+––+– –
Brain tumors
H4 +–+––+– –
Hs683 ++++–+– –
SW1088 + – + – – + – –
SW1783 + – + – – + (+) –
T98G +–+(+)–+(+) (+)
U87 + – + (+) – + (+) –
U118 +(+)+––+– –
U373 +–+––+– –
Miscellaneous
tumor types
Hs294T + – + – – + – –
Table 4: Galectin gene expression by human tumor cell lines
Gal-1 Gal-2 Gal-3 Gal-4 Gal-7 Gal-8
Gal-9
a
(A)
Gal-9
a
(B)
30
Galectin gene expression was determined by RT-PCR analysis as described in Materials and Methods.
Some cell lines did not consistently produce positive signals probably due to low-abundance of the specific
transcript which is indicated by (+).
a
Transcript A represents an isoform, transcript B the expected frag-
ment, data on galectin-9 expression in colon cancer cell lines from Lahm et al. (2000).
ACHN + – + – – + – –
SW13 –––––+– –
DU145 +–+––+– –
LNCaP –––––+– –
PC-3 +–+––+– –
NIH-OVCAR3 + – + – – + (+) (+)
OAW-42 +–+––+– –
M07e +––––+(+) (+)
TF-1 + – (+) – – + (+) (+)
THP-1 + + + (+) – + – (+)
293 +–+–++– –
Table 4: Galectin gene expression by human tumor cell lines
Gal-1 Gal-2 Gal-3 Gal-4 Gal-7 Gal-8
Gal-9
a
(A)
Gal-9
a
(B)
