2. 98 Ringel et al.
INTRODUCTION
Altered expression of cell-surface molecules and the overexpression of growth
factors and their receptors may be involved in malignant progression and metastasis
of human exocrine pancreatic cancer. It has been reported that such growth factors
as basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β1, and
epidermal growth factor (EGF) are abundantly expressed in pancreatic cancer [1– 3].
These growth factors may also be involved in the regulation of adhesion molecules
supposed to be involved in invasion and metastasis.
CD44 is a transmembrane glycoprotein that is implicated in wide variety of
functional roles including cell-cell and cell-matrix interactions. It mediates cell dif-
ferentiation, T-cell activation, cell migration, and metastasis [4,5]. The human CD44
gene is encoded by a gene containing 20 exons. The CD44st standard molecule ex-
presses ten exons, whereas the remaining so-called variant (v) exons can be alterna-
tively spliced in more than 30 different combinations. These variant exons are
expressed in the CD44 variant isoforms [4,5]. The alternative splicing as well as
different degrees of glycosylation result in a multitude of protein isoforms.
Variant isoforms of CD44 were recently described to determine the metastatic
potential of cancer cells. In a rat pancreatic cancer model, the transfection of CD44v6
led to metastatic behavior of the non-metastatic parental pancreatic cancer cell line [6].
CD44 expression has been analyzed in a broad of tissue and cancer types [7–
12]. Different CD44variant isoforms are considered to play a critical role in growth
and metastatic behavior of various tumors [13]. In addition, in some cancer types, a
correlation between CD44v expression and clinical prognosis was observed [7,12].
However, results in different cancer types are controversial [13,14].
The tissue- and cancer-specific expression pattern of CD44 molecules may be the
result of specific splicing and regulation processes. The influence of cytokines like
interferon (IFN) γ or tumor necrosis factor (TNF) α on the CD44 surface expression
has been demonstrated in epithelial kidney and lung cancer cell systems [15]. The
“standard“ form of CD44 [CD44st) has been found to be overexpressed in pancreatic
tumors (16, 17). Other reported that CD44v5 and CD44v6 are newly expressed in
human pancreatic carcinoma and may play role in tumor cell invasion and metastasis
(18, 19, 20). However, the data concerning the expression of CD44st and CD44v
isoforms in pancreatic cancer are somehow contradictory (20, 21, 22, 23). Little is
known about the regulation of the CD44 expression in human pancreatic cancer cells.
To clarify the CD44 protein expression, we investigated different human pan-
creatic cancer cell lines and two SV40 transfected human ductal cell lines using
immunocytochemistry and flow cytometry. Besides the semi-quantitative protein ex-
pression analysis of CD44st and CD44v in these cell lines, we evaluated the effects
of bFGF, TGF-β1, EGF, IFNγ, and TNFα on the expression of the CD44st molecule
and the CD44 variant v5 and v6.
MATERIALS AND METHODS
Cell Lines
Two human SV40 transfected human pancreatic duct epithelial cell lines (PDEC)
were used: M540 (provided by F. Real, Barcelona) [24] and E4 (established by our
group) [25,26]. The establishment, culture conditions, and cell characterization of
3. CD44 in Pancreatic Cancer Cell Lines 99
the PDEC were published previously [25,26]. A panel of well-characterized human
pancreatic cancer cell lines was used; BxPC-3 (well differentiated) and Panc-1 (poorly
differentiated) were purchased from the American Type Culture Collection (ATCC;
Rockville, MD). The cell line PaCa-44 (poorly differentiated) was a gift from Dr. J.
Mollenhauer (Rush University, Chicago, IL) [27]. The cell lines SW850 and SW979
were obtained from Dr. Kalthoff (University of Kiel, Germany) [28]. All cancer cell
lines were routinely grown in DMEM supplemented with 10% fetal calf serum, 100
U/ml penicillin, and 100 µg/ml streptomycin.
We also analyzed Panc-1cells stable transfected with full-length cDNA of TGF-
β1 under the control of a CMV early promoter (Löhr et al., submitted). This plasmid
also codes for the neo resistance gene, such enabling selection of transfectants with
the antibiotic G418. As control cells we used MOCK transfected Panc-1 cells.
Immunocytochemistry
The following panel of mAbs were used: SFF-2 (CD44st) (Serva, Heidelberg,
Germany), anti CD44v3 (R&D), VFF-8 (CD44v5), VFF-18 (CD44v6), VFF-9
(CD44v7), VFF-17 (CD44v7/8), VFF-16 (CD44v10) (all from Serva), anti-ICAM-1
(Roche, Mannheim, Germany), IgG-control antibodies, and FITC-conjugated anti-
mouse Fab (Sigma Chemical Co., St. Louis, MO).
Expression of CD44st, CD44v5 and CD44v6 was determined by immunocy-
tochemistry performed by the three-step method with HRP-conjugated secondary
and tertiary antibodies as described [26]. All primary antibodies and control antibod-
ies were used in dilutions ranging from 1:10–1:100.
FACS Analysis
For flow cytometric analysis of CD44st, CD44 variants v3, v4, v5, v6, v7, v7/8,
v10, and ICAM-1 cells were detached with 5mM EDTA solution and washed with
PBS; 105
cells were incubated with primary antibodies or with an isotype-matched
control mAb for 30 min at 4°C. Then, the cells were washed once and incubated
with 100 ml FITC-conjugated secondary antibody for 30 min at 4°C. The determina-
tion of the surface expression was performed by measuring 10,000 cells of each
sample in a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) by
using FACScan Research software [29].
Growth Factor Stimulation
For stimulation experiments Panc-1 cells (106
) were grown in culture dishes
with DMEM supplemented with 10% FCS for 1 day. Thereafter, the cells were washed
and refed with DMEM without FCS containing bFGF (1, 5, 10, and 100 ng/ml).
After 24, 48, and 72 h, the cells were detached with 5 mM EDTA. Cells were incu-
bated with specific mAbs and stained with a FITC-conjugated secondary antibody as
described above. The effects of IFNγ, TNFα, and EGF were determined by incuba-
tion with IFNγ (250 and 500 U/ml), TNFα (100 and 200 U/ml), and EGF (10 and 20
ng/ml) for 24 and 48 h. For stimulation experiments with TGF-β1, native Panc-1,
and TGF-β1 transfected Panc-1 cells (106
) (transfected cells grown in the presence
of 400 µg/ml G418) were grown in culture dishes with DMEM (without FCS) supple-
mented with 1 or 10 ng/ml TGF-β1 for 48 h. The CD44st and CD44 variant exon v5
and v6 expression was analyzed by FACScan flow cytometer [29]. All experiments
were done three times independently.
4. 100 Ringel et al.
RESULTS
Immuncytochemical Staining and Flow Cytometric Analysis of
CD44st and CD44v
CD44st, v5, and v6 expression may play a critical role in pancreatic cancer.
Therefore, the expression patterns of CD44st and of the variant exons v5 and v6
were examined by immunocytochemistry in the pancreatic cancer cell lines BxPC-3
(well differentiated), Panc-1 (poorly differentiated), and the SV40 large T transfected
human pancreatic duct cell lines M450 and E4. Both cancer cell lines displayed mod-
erate staining for CD44st (mAb SFF-2), CD44v5 (mAb VFF-8), and CD44v6 (mAb
VFF18) (Fig. 1). In contrast, in the two human SV40 transfected pancreatic duct cell
lines, M540 and E4, CD44st was expressed only weakly until reaching a moderate
level (Fig. 1). The two CD44 variants exhibited only a very weak staining pattern in
M540 and E4 cells (Fig. 1).
We used flow cytometry to quantify the surface expression of CD44st, v5, and
v6 as well as to analyze the expression of the CD44 variants v3, v4, v7, v7/8, and
v10 in the pancreatic cancer cell lines. Besides BxPC-3 and Panc-1, the pancreatic
tumor cell lines PaCa44 (poorly differentiated), SW979, and SW850 were investi-
gated. The cell lines SW979 and SW850 differ from the other cell lines, because
they have no known mutations in p53 and Ki-ras genes, which have been described
as occurring with high frequency in pancreatic adenocarcinoma cells [28].
Flow Cytomentry demonstrated CD44st, v5, and v6 in the cell lines BxPC-1
and Panc-1, (Fig. 2, Table 1) that further supported the immunochemical findings.
Fig. 1. Immunocytochemistry for CD 44 and variants. Immunocytochemical staining of the human
pancreatic carcinoma cell line BxPC-3 (A,B) and the human SV40 immortalized duct cells E4 (C,D)
using the antibodies SFF-2 against CD44st (A,C) and VFF-18 against CD44v6 (B,D). Note the intense
staining of CD44st and CD44v6 in the BxPC-3 cells (A,B) and the weak expression in the ductal cell
line E4 (C,D).
5. CD44 in Pancreatic Cancer Cell Lines 101
Fig. 2. FACS analysis histograms for CD44st and CD44 variants. The cell line Panc-1 showed a
staining for CD44st, v3, v4, v5, v6, and v7. The closed line shows the CD44 antigen and the black
diagram is the IgG isotype matched control mAb.
Our analyses revealed that all investigated cell lines, independent of the differentia-
tion grade and the mutations in the p53 and Ki-ras genes, were reactive for CD44st,
CD44v3, CD44v4, CD44v5, and CD44v6. The quantitative level of CD44st and v3,
v4, v5, and v6 variants varied little between the individual cell lines. The well-differ-
6. 102Ringeletal.
TABLE I. Expression of CD44st and Variants in the Different Pancreatic Cancer Cell Lines Analyzed by
FACS-Analyses*
CD44st CD44v3 CD44v4 CD44v5 CD44v6 CD44v7 CD44v7/8 CD44v10 ICAM-1
BxPC-3 335 225 230 200 232 46 93 110 410
Panc-1 158 171 165 173 158 155 0 0 324
PaCa44 165 156 167 175 225 67 85 99 305
SW850 225 129 99 155 183 65 0 0 320
SW979 236 143 132 204 212 60 0 0 314
*The fluorescence mean channels represent the fluorescence intensity of the CD44 expression. The relative fluorescence
mean channels for CD44 are subtracted from the fluorescence mean channels of the control antibody (IgG). The well
differentiated cell line BxPC-3 showed an augmented CD44st staining in comparison to the CD44 variants. ICAM-1 was
analyzed as a control. In all cell lines the ICAM-1 expression was higher then the CD44 staining levels. These are the
representative results of one of three independent FACS analyses for each cell line.
7. CD44 in Pancreatic Cancer Cell Lines 103
entiated cell line BxPC-3 showed an augmented CD44st expression in comparison
to the CD44 variant exons. Only two pancreatic cancer cell lines, the cell line BxPC-
3 and the poorly differentiated cell line PaCa44, expressed variant exon v10 weakly.
Furthermore, the brightness of staining with the CD44v7 VFF-7 mAb was generally
low. The differences in the observed pattern between the CD44v7 VFF-7 mAb and
the CD44v7/8 VFF-17 mAb may be determined by the low expression grade, the
different binding epitopes, and the binding affinities.
The cell surface molecule ICAM-1 has been described to be overexpressed in
pancreatic cancer cells [29]. Therefore, we used this molecule as a control for the
flow cytometric analyses. However, in all cell lines the fluorescence intensity for
CD44st and the variant exons revealed by flow cytometry was significantly lower
than the expression level of ICAM-1 (Table I).
Regulation of Expression of CD44st and CD44 Variant Exons
Growth factors like bFGF and TGF-β1 and cytokines like TNFα may directly
and indirectly affect pancreatic carcinogenesis. Therefore, we investigated the pos-
sible regulation effects of bFGF, TGF-β1, EGF, TNFα, and IFNγ on the surface
expression of CD44st and CD44v5, and v6 in the human pancreatic cell line Panc-1.
IFNγ treatment results in a slight reduction of the CD44v6 surface expression,
whereas CD44st and CD44v5 were unchanged (Fig. 3). These results were indepen-
dent of the IFNγ concentration. Panc-1 cells treated with bFGF, EGF, and Tα showed
no changes in the surface expression of CD44st, v5, and v6. Furthermore, incubation
with TGF-β1 did not affect the expression (data not shown). In addition, we mea-
sured the CD44 protein expression in TGF-β1 transfected Panc-1 cells in compari-
son to MOCK transfected control cells to investigate the possible effects of a TGF-β1
overexpression. No significant difference was found in TGF-β1 transfected Panc-1
cells compared with the control cells (data not shown).
Fig. 3. CD44 expression in response to IFNγ treatment. Expression of CD44st, CD44v5, and CD44v6
on Panc-1 cells using flow cytometry after incubation with IFNγ (250 U/ml; 500 U/ml) in DMEM
without FCS for 48 h. The fluorescence mean channels for CD44v6 as value for the fluorescence
intensity were decreased after incubation with IFNγ independent of the concentration. This figure shows
the fluorescence mean channels without the reduction of the fluorescence mean channels of the control
antibody. The data represents the mean ± SD of three experiments. w/o = without IFN.
8. 104 Ringel et al.
DISCUSSION
Altered expression of adhesion molecules on the surface of cancer cells has
been shown to play an important role in cancer growth behavior and in cell migra-
tion, invasion, and metastasis [30,31]. CD44 isoforms are known to be involved in
various biological processes affecting tumor behavior. These include invasion, me-
tastasis, angiogenesis, cell proliferation, and apoptosis [4,5,32–34]. Furthermore, ex-
pression of CD44 molecules, especially CD44 variant forms, was found to be
correlated with the clinical outcome of various cancer types [7,12].
Recently, a significant correlation was reported between CD44v6 and a decreased
overall survival in pancreatic cancer [22]. Some investigators have found that CD44v5
and CD44v6 are expressed in human pancreatic adenocarcinoma cells but not in
normal pancreatic tissue [20,23]. In one study involving a SCID mice metastasizing
model, CD44st was found to be downregulated in metastatic cells while a large frac-
tion of pancreatic cancer cells expressed on membrane the splice variants v5/v6 [18].
However, another report showed that CD44v6 is detectable in normal pancreatic tis-
sue as well as in human pancreatic cancer samples [20]. The reasons for the discrep-
ancies observed could be related to the different techniques used for the expression
analyses of CD44. In most studies, the CD44 expression has been explored by RT-
PCR and/or immunohistochemistry [20–23]. Because different studies are performed
with different techniques, antibodies, primers, and probes, it is very difficult to com-
pare their results. Moreover, there is some evidence that not all CD44 isoforms de-
tectable on RNA level are translated to the protein level [20]. Furthermore, the different
results by using immunocyto- and immunohisto-chemistry may be caused by using
different CD44 antibodies. Besides the different splicing forms of CD44 molecules,
their expression levels may be important for the pathogenic behavior of human pan-
creatic cancer cells.
We performed these studies to determine the protein expression of CD44st and
CD44 variants, especially v5 and v6, in pancreatic cancer cell lines using immunocy-
tochemistry and, for quantification, flow cytometry. We observed a staining for CD44st,
CD44v5, and v6 as well as for CD44v3 and v4 in all investigated cancer cell lines.
These results are supported by data published by other groups [19,20]. The surface
level of CD44 forms was generally lower compared to the adhesion molecule ICAM-
1, which is proposed to be highly expressed in pancreatic cancer cells [30].
In addition, we investigated the expression of CD44st, v5, and v6 in two SV40
large T antigen transfected non-tumorigenic human ductal cell lines. Both cell lines
showed only a weak staining for CD44st, CD44v5, and v6 compared to the stronger
expression in the pancreatic cancer cell lines. These findings are interesting in the
light of a published study that describes the expression of CD44v5 and v6 in almost
all duct cell carcinomas and invasive cancers [19]. We also investigated possible
regulation mechanism, which might influence the surface expression of CD44st,
CD44v5, and v6. To date, little information is available about the regulation of CD44
in pancreatic cancer. In CAPAN-1 cells, a reduction of CD44-positive cells was de-
tected after incubation with interleukin 1β and interleukin 1β [36]. In this study, we
investigated the effects of bFGF, TGF-β1, EGF, TNFα, and IFNγ, which are be-
lieved to be involved in carcinogenesis of human pancreatic adenocarcinoma [1,36]
also to affect the CD44 cell surface levels in other cell types [1,2,15]. Only IFN γ
affected the staining intensity by downregulation of CD44v6 in the Panc-1 cell line.
Interestingly, the CD44variant form v5 was not altered. In contrast to the findings on
9. CD44 in Pancreatic Cancer Cell Lines 105
the CAPAN-1 cells, we could not detect an increase of CD44 either after incubation
with external TGF-b1 or after transfection of full length TGF-β1 in Panc-1 cells. The
conflicting data about the TGF-β1 effects may be the results of the use of different
pancreatic cancer cell lines, which are known to respond variably to TGF-β1.
In conclusion, we demonstrated the protein expression of malignancy- related
CD44 variant forms v5 and v6 in all cancer cell lines and only a weak expression in
SV 40 large T transfected human ductal cells. According to our FACS analysis, the
staining level for CD44st and variants in the cancer cells was lower than the ICAM-
1 expression. Furthermore, the regulation of CD44 expression in pancreatic cancer
cells seems to differ from other cell types.
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![98 Ringel et al.
INTRODUCTION
Altered expression of cell-surface molecules and the overexpression of growth
factors and their receptors may be involved in malignant progression and metastasis
of human exocrine pancreatic cancer. It has been reported that such growth factors
as basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β1, and
epidermal growth factor (EGF) are abundantly expressed in pancreatic cancer [1– 3].
These growth factors may also be involved in the regulation of adhesion molecules
supposed to be involved in invasion and metastasis.
CD44 is a transmembrane glycoprotein that is implicated in wide variety of
functional roles including cell-cell and cell-matrix interactions. It mediates cell dif-
ferentiation, T-cell activation, cell migration, and metastasis [4,5]. The human CD44
gene is encoded by a gene containing 20 exons. The CD44st standard molecule ex-
presses ten exons, whereas the remaining so-called variant (v) exons can be alterna-
tively spliced in more than 30 different combinations. These variant exons are
expressed in the CD44 variant isoforms [4,5]. The alternative splicing as well as
different degrees of glycosylation result in a multitude of protein isoforms.
Variant isoforms of CD44 were recently described to determine the metastatic
potential of cancer cells. In a rat pancreatic cancer model, the transfection of CD44v6
led to metastatic behavior of the non-metastatic parental pancreatic cancer cell line [6].
CD44 expression has been analyzed in a broad of tissue and cancer types [7–
12]. Different CD44variant isoforms are considered to play a critical role in growth
and metastatic behavior of various tumors [13]. In addition, in some cancer types, a
correlation between CD44v expression and clinical prognosis was observed [7,12].
However, results in different cancer types are controversial [13,14].
The tissue- and cancer-specific expression pattern of CD44 molecules may be the
result of specific splicing and regulation processes. The influence of cytokines like
interferon (IFN) γ or tumor necrosis factor (TNF) α on the CD44 surface expression
has been demonstrated in epithelial kidney and lung cancer cell systems [15]. The
“standard“ form of CD44 [CD44st) has been found to be overexpressed in pancreatic
tumors (16, 17). Other reported that CD44v5 and CD44v6 are newly expressed in
human pancreatic carcinoma and may play role in tumor cell invasion and metastasis
(18, 19, 20). However, the data concerning the expression of CD44st and CD44v
isoforms in pancreatic cancer are somehow contradictory (20, 21, 22, 23). Little is
known about the regulation of the CD44 expression in human pancreatic cancer cells.
To clarify the CD44 protein expression, we investigated different human pan-
creatic cancer cell lines and two SV40 transfected human ductal cell lines using
immunocytochemistry and flow cytometry. Besides the semi-quantitative protein ex-
pression analysis of CD44st and CD44v in these cell lines, we evaluated the effects
of bFGF, TGF-β1, EGF, IFNγ, and TNFα on the expression of the CD44st molecule
and the CD44 variant v5 and v6.
MATERIALS AND METHODS
Cell Lines
Two human SV40 transfected human pancreatic duct epithelial cell lines (PDEC)
were used: M540 (provided by F. Real, Barcelona) [24] and E4 (established by our
group) [25,26]. The establishment, culture conditions, and cell characterization of](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)