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Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
Int. J. Cancer: 80, 791–795 (1999)
r 1999 Wiley-Liss, Inc.
INTEGRIN ␣6␤1 ROLE IN METASTATIC BEHAVIOR
OF HUMAN PANCREATIC CARCINOMA CELLS
Roger VOGELMANN1, Ernst D. KREUSER2, Guido ADLER1 and Manfred P. LUTZ1*
1Department of Internal Medicine I, University of Ulm, Ulm, Germany
2Department of Hematology and Oncology, University Medical Center Benjamin Franklin, Free University, Berlin, Germany
The factors that determine the metastatic behavior of
pancreatic tumor cells are incompletely understood. In this
study, we first demonstrate differences in adhesion properties, integrin expression and in vivo integrin function in the
metastatic tumor cell line PaTu 8988s compared with the
non-metastatic cell line PaTu 8988t. Both cell lines were
derived from the same original tumor and exhibit identical
genetic fingerprints. Using in vitro adhesion assays performed
on purified extracellular matrix components, adhesion of
PaTu 8988s cells was significantly increased on the basal
membrane component laminin and decreased on the interstitial matrix protein fibronectin compared to PaTu 8988t cells.
By immunocytochemistry and flow cytometry, and in correspondence with their adhesive properties, the metastatic
PaTu 8988s cells did express a distinct pattern of integrin
subunits. Laminin-binding integrins ␣6 and ␤4 were overexpressed in PaTu 8988s cells. Fibronectin-binding ␣5 integrins
were present at higher levels in the non-metastatic PaTu
8988t cells, whereas the ␤1 subunit expression did not differ.
Adhesion to laminin or fibronectin was specific and was
mediated via integrins ␣6␤1 and ␣5␤1, respectively. In addition, metastasis formation in vivo after injection of cells into
the tail vein of nude mice was inhibited by preincubation of
PaTu 8988s cells with antibodies directed against the integrin
␣6 or ␤1. We conclude that ␣6␤1 integrins are overexpressed
and functionally active in metastatic human pancreatic carcinoma cells, and participate in metastasis formation probably
through binding to the basal membrane component laminin.
Int. J. Cancer 80:791–795, 1999.
r 1999 Wiley-Liss, Inc.
Pancreatic cancer is a major cause of cancer-related deaths in
Western countries. Despite intensive efforts to improve therapy of
advanced disease, palliation remains unsatisfactory and most
patients die within months due to rapid locoregional growth of the
tumor or early metastasis formation. The biological characteristics
that are responsible for the aggressive behavior of these tumors are
not well understood.
Tumor metastasis is considered to be a multistep process. During
this process, tumor cells must detach from the primary tumor,
migrate through adjacent tissues and enter the circulation where
they face the host defenses. In addition, metastasizing cells need to
arrest at a distant vascular site, cross the basal membrane, which
functions as a barrier between circulation and target organ, and be
able to colonize and grow under these novel conditions. Many of
these steps require changes in the adhesive properties of cells to the
extracellular matrix (Heino, 1996).
Cell adhesion to extracellular matrix is mediated at least in part
by the integrin family of transmembrane receptor proteins. Integrins are dimeric proteins composed of non-covalently associated
␣ and ␤ subunits. Theoretically, the 8 known ␤ subunits can
combine with one of at least 16 ␣ subunits, resulting in integrin
heterodimers with distinct adhesion properties. The integrins are
divided into subgroups depending on their binding preferences to
extracellular matrix proteins or to cell surface molecules. To date,
22 different ␣␤ complexes have been described, with 15 of these
acting as receptors for fibrous protein components of the extracellular matrix (Heino, 1996). These matrix proteins are classified into
structural proteins (e.g., collagen or elastin) or proteins with mainly
adhesive properties (e.g., fibronectin or laminin). The adhesion of
cells to interstitial matrix is mediated by fibronectin together with
collagen type I as structural component, whereas laminin is the
predominant adhesive protein in the basal membrane together with
collagen type IV as a backbone. Decreased expression of fibronectinbinding integrins or increased expression of laminin-binding
proteins correlates with aggressive growth and with the ability to
metastasize in several tumor tissues (Shaw et al., 1996; Schreiner et
al., 1991; Giancotti and Ruoslahti, 1990; Varner et al., 1995).
In the pancreas, the expression of several integrin subunits has
been described. These include the fibronectin-binding integrin ␣5;
the laminin-binding integrins ␣2, ␣3 and ␣6; and the vitronectinbinding ␣v together with the ␤1, ␤4 and ␤5 subunits. In general,
expression of the fibronectin-binding ␣5 subunit appears to be
decreased in tumor tissues (Hall et al., 1991). Using functional
assays, pancreatic tumor cells can attach to laminin, fibronectin or
collagens (Weinel et al., 1995; Löhr et al., 1996), and adhesion to
laminin appears to be mediated by the laminin-specific integrin
␣6␤1 (Weinel et al., 1995; Rosewicz et al., 1997). Integrin
expression does not correlate with tumor differentiation, and the
number of tumors or cell lines examined has been too small to
define the changes relevant to the growth characteristics or the
metastatic behavior of these cells (Hall et al., 1991; Rosendahl et
al., 1993; Löhr et al., 1996).
Therefore, we used 2 closely related cell lines, both derived from
the same original tumor, but differing in metastatic ability (Elsässer
et al., 1992) to examine adhesion properties, integrin expression
and integrin function. Using expression studies and adhesion
assays on purified matrix components, we could define a metastasisrelated expression pattern of functionally intact integrins with
binding specificity for laminin. In addition, inhibition experiments
with integrin-specific antibodies demonstrated a role for the
laminin-binding integrins ␣6␤1 in experimental in vivo metastasis.
MATERIAL AND METHODS
Cell lines
The human pancreatic carcinoma cell lines PaTu 8988s and PaTu
8988t (Elsässer et al., 1992) were obtained from the Deutsche
Sammlung für Mikroorganismen (Braunschweig, Germany). Both
cell lines express a unique fingerprint profile when using the (gtg)5
and pYNH24 probes. Cells were maintained under standard culture
conditions in Dulbecco’s modified Eagle’s medium (DMEM) with
high glucose, 10% fetal calf serum (FCS) and 1% antibiotics/
antimycotics (GIBCO, Eggenstein, Germany).
Antibodies, proteins and peptides
Flow cytometry and immunocytochemistry were performed
using monoclonal antibodies (MAbs) specific for the integrins ␣1
(clone TS2/7; Serotec, Oxford, UK), ␣2 (Gi9), ␣3 (PiB5), ␣4,
(HP2/1), ␣5 (SAM-1), ␣6 (GoH3), ␣v (AMF7), ␤1 (K20), ␤2
(BL5), ␤3 (SZ.21), ␤4 (3E1) and ␣v␤5 (P1F6; all from Dianova,
Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: Lu
441/2-1.
*Correspondence to: Department of Internal Medicine I, University of
Ulm, 89081 Ulm, Germany. Fax: (49) 731-502-4323.
E-mail: [email protected]
Received 29 September 1998
792
VOGELMANN ET AL.
Hamburg, Germany), and directed against IgG1 (MOPC-21; Sigma,
Deisenhofen, Germany). Collagen type I, collagen type IV and
fibronectin were from Sigma, and laminin was obtained from
Biomol (Hamburg, Germany). The specificity of cell adhesion was
tested using the MAbs described above, except for ␤1 integrin
(DE9; Biomol). The peptides RGD, GRGDS, GRADSPK and
YIGSR were purchased from Bachem (Heidelberg, Germany).
Purified rat IgG and purified mouse IgG for metastasis assays were
from Sigma and Dianova, respectively. All other reagents were of
analytical grade.
Flow cytometry
As described in detail by Herzberg et al. (1996), trypsinized cells
were washed once and resuspended at a concentration of 1 ⫻ 107
cells/ml in calcium and magnesium-free phosphate-buffered saline
(PBS) containing 5% FCS. After incubation for 30 min with
saturating concentrations of antibodies at 4°C, cells were washed
once in PBS/5% FCS. Labeling was carried out with fluorescein
isothiocyanate (FITC)-conjugated F(ab8)2 fragments for 30 min at
4°C. After an additional washing step, cells were analyzed using a
FACScan (Becton-Dickinson, Heidelberg, Germany) with LYSIS
II software. Antibodies against IgG1 were used as a basal control. A
live gate was set by staining with 2 µg/ml propidium iodide for 5
min. The threshold level for significance of integrin expression was
arbitrarily set at channel 50.
Adhesion assay
Semiconfluent cells in culture were washed with PBS containing
1 mM EDTA and were detached by short treatment with PBS/0.2%
EDTA/0.5% trypsin. After 2 washing steps in Tris-buffered saline
containing 0.01% (w/v) soybean trypsin inhibitor (Boehringer
Mannheim, Germany), cells were resuspended to a density of
50,000 cells/100 µl Tris-buffered saline containing 0.1% heatdenatured bovine serum albumin (BSA), 2 mM glucose and 2 mM
MgCl2 (TBS). Extracellular matrix protein-covered cell culture
plates were prepared by incubation of sterile, non-tissue culture
96-well flat-bottom plates (Greiner, Frickenhausen, Germany) with
the indicated concentrations of matrix proteins overnight at room
temperature. Unspecific adhesion was blocked by incubation with
1% BSA (low endotoxin; Sigma) in PBS for 60 min at room
temperature. For adhesion assays, 100 µl of cell suspension was
added to each well and adhesion was allowed to proceed at
37°C/5% CO2 for the indicated time periods. Non-adherent cells
were removed by 2 careful washing steps in PBS. The fraction of
adherent cells was determined by staining with sulforhodamine B
(Skehan et al., 1990). Briefly, cells were fixed by adding 125 µl of
ice-cold trichloroacetic acid (TCA) solution (10% in DMEM) for
60 min. After 5 washing steps with H2O, the cells were stained with
0.4% sulforhodamine B in 1% acetic acid for 30 min. Following 4
additional washing steps in 1% acetic acid, the absorbed dyes were
redissolved in 10 mM Tris base, pH 10.5, and the extinction was
measured at 577 nm. Extinction did correlate with the cell number
in a linear fashion under the assay conditions used. To calculate the
fraction of adhering cells, the extinctions obtained in the absence of
cells were subtracted, and values were expressed as percentage of
the extinctions obtained without washing steps.
For inhibition experiments, detached cells were preincubated in
TBS with or without the indicated concentrations of MAbs,
peptides or laminin for 60 min at 37°C under constant rotation, and
adhesion assays were then performed as described above. In
preliminary experiments, increasing concentrations were tested to
determine the concentrations necessary for maximum inhibition.
Adhesion was expressed as percent of extinction measured after
preincubation with TBS alone.
Metastasis assay
To determine their metastatic potential, cells were prepared as
described above and were suspended in cell culture medium; 100 µl
of the cell suspension containing 2 ⫻ 106 cells was injected into the
tail vein of NMRI nu-nu mice. After 4 weeks, the animals were
sacrificed and both lungs were excised. Lung tissue was fixed in 4%
formaldehyde. The total number of colonies were blindly counted
in serial sections after staining with hematoxylin-eosin (H&E). The
animal care protocol and the experimental design were approved
by a governmental animal care review committee.
Statistics
The mean values and standard errors were calculated out of at
least 3 experiments, each performed at least in quadruplicate. For
statistical analysis, the Wilcoxon non-parametric test was used, and
p ⬍ 0.05 was considered significant.
RESULTS
Adhesion assays
In vitro adhesion was examined on purified extracellular matrix
proteins. The results of time-dependent adhesion on the major
interstitial matrix components collagen type I and fibronectin and
the basal membrane components collagen type IV and laminin are
shown in Figure 1. Assay conditions had been validated before by
using increasing amounts of extracellular matrix proteins and by
varying the divalent cation concentration. Adhesion did increase
with substrate concentration and reached a plateau above 3.125
µg/ml for collagen I and collagen IV, and above 12.5 µg/ml for
laminin in both cell lines. Maximal adhesion on fibronectin was
observed above 6.25 µg/ml for PaTu 8988s cells and above 12.5
µg/ml for PaTu 8988t cells. Adhesion increased with the concentration of Mg2⫹, reaching its maximum level at 4 mM Mg2⫹ on all 4
substrates. Changing the Ca2⫹ concentration did not influence
maximal adhesion. Therefore, 12.5 µg/ml substrate protein was
used for surface coating in subsequent experiments and a concentration of 2 mM Mg2⫹ was used in the adhesion assays to approximate
physiological conditions.
Maximal adhesion was similar for both cell lines on the
structural matrix proteins collagen types I and IV. In contrast,
binding to the adhesive proteins laminin and fibronectin differed
significantly between cell lines. Maximal adhesion of the metastatic cell line PaTu 8988s on laminin was 2.8 times that of the
non-metastatic cell line PaTu 8988t ( p ⫽ 0.004). In contrast,
adhesion on fibronectin was 8.1 times ( p ⫽ 0.03) higher for PaTu
8988t cells than for PaTu 8988s cells (Fig. 1).
Expression of integrin subunits
Integrin subunit expression in the metastatic cell line PaTu 8988s
and in the non-metastastic cell line PaTu 8988t was quantified by
flow cytometry after immunocytochemical staining (Fig. 2).
FIGURE 1 – Adhesion of PaTu 8988s (䊊) and PaTu 8988t (䊏) cells
on purified extracellular matrix proteins. Shown are the fractions of
adhering cells after various time periods on the interstitial matrix
proteins fibronectin (c) and collagen type I (a), and on the basal
membrane proteins laminin (d) and collagen type IV (b). Values are
expressed as means ⫾ SEM of at least 3 independent experiments.
INTEGRIN ␣6␤1 AND PANCREATIC CARCINOMA
793
FIGURE 2 – Integrin subunit expression in pancreatic carcinoma cell
lines PaTu 8988s (white bars) and PaTu 8988t (black bars). Cells were
labeled with integrin-specific antibodies and analyzed by flow cytometry. Values are expressed as means ⫾ SEM of 2 independent
determinations.
Expression of the ␤1 subunit, and low expression levels of the
␤4 and ␤5 subunits were demonstrated in both cell lines. The ␤1
subunit that is the most ubiquitous integrin ␤ subunit in epithelial
cells and is able to associate with most ␣ subunits was present in
similar amount in both cell lines. Expression of the ␤4 subunit that
is known to bind to laminin in combination with the ␣6 subunit, as
well as the ␤5 subunit that mediates binding to vitronectin if
dimerized to ␣v (Heino, 1996), was more prominent in the
metastatic cell line PaTu 8988s. Expression of ␤2 and ␤3 subunits
was below the threshold level.
Several ␣ subunits were expressed in both cell lines. In PaTu
8988s cells, expression of ␣1 (preferentially binding collagen type
I and laminin), ␣2 (preferentially binding collagen type VI and
laminin), ␣6 (binding to laminin) and ␣v (binding to fibronectin
with low specificity) was more marked. In contrast, expression
levels of ␣5, which is the main fibronectin-binding subunit, were
elevated in PaTu 8988t cells. Expression of the ␣4 subunit was
below the threshold level in both cell lines.
Specificity of adhesion
To test the specificity of adhesion for the substrates, cells were
preincubated either with laminin or with fibronectin-derived peptides. As illustrated in Figure 3, binding to laminin was inhibited
after preincubation with the soluble laminin molecule (3.125
µg/ml) in PaTu 8988s cells by 47.0 ⫾ 10.2% ( p ⫽ 0.02) and in
PaTu 8988t by 39.5 ⫾ 5.0% ( p ⫽ 0.02). Under identical conditions, the YIGSR peptide (0.5 mM), which represents the laminin
region binding to the 67 kDa laminin receptor (Graf et al., 1987),
had no effect on the adhesion of both cell lines on laminin. Next,
binding to fibronectin was inhibited using known integrin-binding
sequences of the fibronectin molecule (Hardan et al., 1993).
Inhibition by the RGD peptide (0.5 mM) was less effective than
preincubation with the GRGDS peptide (0.5 mM), which reduced
the adhesion of PaTu 8988t on fibronectin to 42.8 ⫾ 8.4%
( p ⫽ 0.02) of controls. In control experiments, the same peptides
did not inhibit adhesion of either cell lines to laminin, and laminin
at a concentration of 3.125 mg/ml, which had been shown to
maximally inhibit binding to laminin, did not decrease adhesion to
fibronectin.
Participation of specific integrin chains in cell adhesion was
examined by preincubation of cells with integrin-specific MAbs.
As illustrated, adhesion to laminin in PaTu 8988s cells was
inhibited by 40.2 ⫾ 6.6% ( p ⬍ 0.005) with antibodies against ␤1
(1.25 µg/ml) and by 39.0 ⫾ 11.5% when using anti-␣6 antibodies
(1.25 µg/ml, p ⬍ 0.005), whereas anti-␤4 antibodies (60 µg/ml) did
not have any effect (Fig. 3). Similar results were observed using
PaTu 8988t cells, where anti-␤1 decreased binding to laminin by
48.9 ⫾ 9.3% ( p ⬍ 0.005) and anti-␣6 by 50.0 ⫾ 4.7% ( p ⫽ 0.02).
As expected, antibodies against the fibronectin-specific ␣5 subunit
FIGURE 3 – Inhibition of maximal adhesion of PaTu 8988s cells
(white bars) or PaTu 8988t cells (black bars) on laminin (a) or
fibronectin (b). Cells were preincubated with the indicated concentrations of proteins, peptides or antibodies for 60 min and were allowed to
adhere for 60 min. Shown are the fractions of adhering cells in the
presence of inhibitors relative to controls. Values are expressed as
means ⫾ SEM of at least 3 independent experiments.
(1.25 µg/ml) as well as purified polyclonal mouse IgG (10 mM) did
not decrease attachment to laminin. The same anti-␣5 antibody
decreased binding of PaTu 8988s and PaTu 8988t cells to
fibronectin by 52.0 ⫾ 5.4% ( p ⬍ 0.005) and by 45.0 ⫾ 16.6% (not
significant), respectively. Fibronectin binding was inhibited with
antibodies directed against the ␤1 subunit (PaTu 8988s:
32.2 ⫾ 6.4%, p ⬍ 0.005; PaTu 8988t: 48.6 ⫾ 4.9%, p ⬍ 0.005),
whereas antibodies against ␤4 and ␣6 as well as IgG did not change
adhesion significantly. Of note, this occurred even though ␤4
integrin subunits were present in our cell lines on the plasma
membrane (data not shown) and ␤4 may associate with ␣6 in
pancreatic tumor cells (Weinel et al., 1995).
Metastasis formation
In vivo integrin function was examined using a mouse model of
metastasis formation in lung tissue. Injection of PaTu 8988s cells
into the tail vein of NMRI nu-nu mice led to growth of 7.6 ⫾ 2.7
colonies/animal (n ⫽ 20, range 0–44) metastases in both lungs. In
contrast, and as reported originally (Elsässer et al., 1992), PaTu
8988t cells did not colonize lung tissue (n ⫽ 13). As shown above,
PaTu 8988s cells when compared with PaTu 8988t cells adhered
more strongly to laminin and expressed more laminin-specific
integrin subunits; in addition, binding to laminin was decreased by
antibodies against the integrin subunits ␣6 and ␤1. To test the
potential role of these laminin-binding integrins for metastasis
formation, 8988s cells were preincubated with integrin-directed
antibodies before injection. As shown in Figure 4, preincubation
with the antibody directed against the laminin-binding ␣6 integrin
significantly decreased lung colony formation to 0.6 ⫾ 0.2
colonies/animal (n ⫽ 13, p ⫽ 0.02, compared to control), whereas
antibodies directed against the ubiquitous ␤1 integrin subunit led to
a reduction to 1.1 ⫾ 0.4 lung colonies/animal (n ⫽ 8, p ⫽ 0.2). In
control experiments, immunoglobulin controls for the anti-␣6 (rat
VOGELMANN ET AL.
794
FIGURE 4 – Metastasis formation in lung tissue. Cells were injected
into the tail vein of NMRI nu-nu mice after preincubation for 60 min in
buffer alone or buffer with antibodies against integrin ␣6, integrin ␤1 or
immunoglobulin controls. The number of metastases was counted after
4 weeks by histological examination. Values are expressed as means ⫾
SEM.
IgG, n ⫽ 9) and anti-␤1 (mouse IgG, n ⫽ 5) antibodies had no
effect on metastasis formation.
DISCUSSION
In this study, we have demonstrated that PaTu 8988s, a
metastasizing human pancreatic carcinoma cell line, preferentially
adheres to laminin and expresses a different pattern of integrin
subunits than its twin cell line PaTu 8988t, which was originally
derived from the same tumor tissue but lacks metastatic potential
(Elsässer et al., 1992). Binding to laminin is thought to represent an
essential step in metastasis (Iwamoto et al., 1987) and might
explain the differences in metastatic capacity observed between the
2 cell lines. We, therefore, first confirmed that adhesion to laminin
was indeed specific and could be inhibited by laminin but not by
fibronectin-derived peptides, and then examined whether integrin
expression could explain the observed differences in adhesion.
Significant differences were observed for several ␣ integrin subunits relevant to binding of cells to laminin or to the interstitial
matrix component fibronectin. The ␣6 subunit, which binds with
high preference to laminin, was over-expressed in the metastatic
cell line 8988s, whereas expression of the ␣5 subunit, which
confers binding to fibronectin, was elevated in the non-metastasizing cell line 8988t. As expected, expression of the ␤1 subunit was
similar in both cell lines, corresponding to its ability to associate
with most ␣ subunits with similar affinity (Heino, 1996). In other
tumor tissues, e.g., prostate cancer (Rabinovitz et al., 1995) or
malignant melanoma (Danen et al., 1993), expression of the ␣6
integrin correlates with the ability of tumor cells to metastasize.
The function of this integrin chain appears to center around
attachment to or invasion through the basal membrane (Heino,
1996), which is supported by in vitro experiments where overexpression of ␣6␤1 integrins in chemically transformed human osteosarcoma cells correlated with their ability to migrate through the
reconstituted basal membrane Matrigel (Dedhar and Saulnier,
1990), and where invasion into Matrigel was inhibited by antibodies against the ␣6 subunit in fibrosarcoma cells (Ramos et al.,
1991), prostate carcinoma (Rabinovitz et al., 1995) and pancreatic
carcinoma cell lines (Weinel et al., 1995). In addition, inhibition of
␣6 integrin function may be one of the mechanisms by which
transfection of breast carcinoma cells with a dominant negative
integrin ␤4 chain inhibits their ability to adhere and migrate on
laminin in vitro (Shaw et al., 1996). In our model, the relative
overexpression of ␣6 integrins in the metastatic PaTu 8988s cell
line is consistent with the hypothesis that integrin-mediated
laminin binding may be important for metastasis of pancreatic
tumor cells.
Cells can bind to laminin through a variety of laminin receptors,
which include the integrins ␣6␤1 and ␣6␤4, but also the 67 kDa
laminin receptor. In our experiments, adhesion was inhibited by
antibodies against the ␣6 and the ␤1 integrin chains, but not by
antibodies directed against the ␤4 integrin subunit or by the peptide
YIGSR, which represents the laminin-binding sequence of the 67
kDa laminin receptor and which competitively inhibits binding
through this mechanism (Graf et al., 1987). Therefore, similar to 5
other pancreatic carcinoma cell lines (Weinel et al., 1995; Rosewicz
et al., 1997), binding of PaTu 8988s cells to laminin was mediated
by the integrin ␣6␤1, and not via ␣6␤4 integrin complexes or the
67 kDa laminin receptor.
To confirm the actual role of the integrin ␣6␤1 for in vivo
metastasis, we did inhibit their function by incubation of PaTu
8988s cells with specific antibodies and could significantly decrease lung colonization in a nude mouse metastasis model. A
similar mechanism has been described for mouse melanoma cells,
where integrin ␣6 antibodies inhibit allogeneic metastasis formation when injected before or simultaneously with the tumor cells, or
when cells had been precoated with antibodies (Ruiz et al., 1993).
However, adhesion of melanoma cells to laminin as well as
metastasis formation can be inhibited by the YIGSR sequence,
which binds to the 67 kDa laminin receptor (Iwamoto et al., 1987).
The same peptide does not inhibit adhesion of PaTu 8988s cells or
other pancreatic tumor cells to laminin (Weinel et al., 1995) to
further strengthen the central position of the ␣6␤1 integrin for
pancreatic cancer cell metastasis.
We conclude that overexpression of ␣6␤1 integrins is one of the
determinants of metastasis formation in pancreatic carcinoma.
Therefore, the diagnostic evaluation of ␣6␤1 integrin expression
might provide valuable prognostic information to establish riskadapted treatment strategies. In addition, inhibition of integrin
function might open ways to novel therapeutic approaches.
ACKNOWLEDGEMENTS
We thank Mrs. F. Genze and Mrs. E. Wolf-Hieber for expert
technical assistance.
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