Production of liquid fuel intermediates from furfural via aldol condensation over La2O2CO3-ZnO-Al2O3 catalyst

Article abstract of DOI:10.1016/j.catcom.2020.106207

Aldol condensation of furfural with acetone over basic catalysts allows the production of furanic adducts 4-(2-furyl)-3-buten-2-one (FAc, C8) and 1,5-di-2-furanyl-1,4-pentadien-3-one (F₂Ac, C13)) that can be transformed into high-quality diesel

Full text of DOI:10.1016/j.catcom.2020.106207

Drebrin, an Actin-Binding, Cell-Type Characteristic Protein:
Induction and Localization in Epithelial Skin Tumors and
Cultured Keratinocytes
Ã
Ã
Wiebke K. Peitsch, w Ilse Hofmann,w Jutta Bulkescher, w Michaela Hergt,w Herbert Spring,z Uwe Bleyl,y
Ã
Ã
Sergij Goerdt, and Werner W. Frankew
Department of Dermatology, Medical Center Mannheim, University of Heidelberg, Mannheim, Germany; wDivision of Cell Biology, German Cancer Research
Center, Heidelberg, Germany; zBiomedical Structure Analysis Group, German Cancer Research Center, Heidelberg, Germany; yDepartment of Pathology, Medical
Center, Mannheim University of Heidelberg, Mannheim, Germany
Isoform E2 of drebrin, an actin-binding protein originally identified in neuronal cells, has recently been identified in
diverse non-neuronal cells, mostly in association with cell processes and intercellular junctions. Here, we report on
the presence of drebrin in normal human skin, epithelial skin cancers, and cultured keratinocytes. Keratinocytes of
normal epidermis contain almost no drebrin but the protein is readily seen in hair follicles. By immunohistochem-
istry and immunoblot, basal cell carcinomas (BCC) are rich in drebrin, and confocal laser scanning and immuno-
electron microscopy show accumulation at adhering junctions, in co-localization with actin and partially with
plaque proteins. In squamous cell carcinomas, keratoacanthomas, and in epidermal precancers, drebrin is het-
erogeneously distributed, appearing as mosaics. Primary keratinocyte cultures contain significant amounts of
drebrin enriched at adhering junctions. When epithelium-derived cells devoid of drebrin are transfected with
drebrin-enhanced green fluorescent protein, constructs accumulate in the cell periphery, and immunoprecipitation
shows complexes with actin. During epidermal growth factor induced formation of cell processes, drebrin retains
this junction association, as observed by live cell microscopy. Our results suggest novel functions of drebrin such
as an involvement in cell–cell adhesion and tumorigenesis and a potential value in diagnosis of BCC.
Key words: actin-binding protein/basal cell carcinoma/catenin/cell adhesion/skin cancer
J Invest Dermatol 125:761 –774, 2005
Skin carcinomas are the most frequent cancers in the light-
skinned population, with an estimated yearly incidence of
approximately 100 basal cell carcinomas (BCC) and approx-
imately 20 squamous cell carcinomas (SCC) per 100,000 in-
habitants. Important and long-known pathogenetic factors
for skin carcinogenesis include ultraviolet (UV) light, radiation,
and a diversity of chemical carcinogens. Only recently, mo-
lecular pathways leading to development of BCC and SCC
have been elucidated. In inherited BCC such as in Gorlin’s
syndrome and in sporadic BCC, gene mutations of proteins
of the Sonic Hedgehog signal transduction pathway have
been identified that lead to basaloid hyperproliferations and
infiltrative tumors (Hahn et al, 1996; for a recent review see,
e.g., Cohen, 2003). In addition, there is increasing evidence
that defects in cell–cell adhesion might contribute to skin
carcinogenesis. For example, downregulation of E-cadherin,
the major transmembrane protein of adhering junctions in
epithelial cells, has been reported to correlate with deve-
lopment, progression, and metastasis of several kinds of
carcinomas (e.g., Behrens et al, 1989; Shimoyama and
Hirohashi, 1991; Moll et al, 1993; Perl et al, 1998; review:
Cavallaro and Christofori, 2004), including SCC of the skin
(Llorens et al, 1998; Lozano and Cano, 1998; Wu et al, 2000;
Papadavid et al, 2002). Moreover, b-catenin, a constitutive
protein of the cytoplasmic plaque of adhering junctions, is
part of the Wnt signaling pathway, a cascade of reactions
leading to transcription of target genes for proliferation (re-
cently reviewed by Nelson and Nusse, 2004). Specifically in
the skin, activating mutations of b-catenin cause proliferation
of cells of the hair follicle sheath and development of pi-
lomatrixomas (Gat et al, 1998; Chan et al, 1999; Doglioni
et al, 2003), and this protein might also be involved in the
formation of BCC (El-Bahrawy et al, 2003).
In addition to their involvement in tumor development
and progression, proteins of intercellular junctions can also
serve as cell-type ‘‘markers’’ in tumor diagnosis and are
thus of particular value in cases of unknown primary tumors
and for determination of the degree of differentiation. This
has been shown extensively for plaque proteins of desmo-
somes such as desmoplakin (e.g., Franke et al, 1983; Moll
et al, 1986; Jones and Grelling, 1989; review: Garrod, 1995),
the plakophilins (Heid et al, 1994; Mertens et al, 1999), and
the various desmosomal cadherins (Parrish et al, 1986;
Nuber et al, 1995; Sch a¨ fer et al, 1996; for skin tumors see
Abbreviations: BCC, basal cell carcinoma; EGF, epidermal growth
factor; EGFP, enhanced green fluorescent protein; IRS, inner root
sheath; mab, monoclonal antibody; ORS, outer root sheath; PBS,
phosphate-buffered saline; RT, room temperature; SCC, squamous
cell carcinoma
Copyright r 2005 by The Society for Investigative Dermatology, Inc.
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62 PEITSCH ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Krunic et al, 1996; Moll et al, 1997). Similarly, several cell-
type-specific components of adhering junctions, notably
members of the cadherin and the armadillo families of pro-
teins, have been introduced into pathology (for references,
see Mareel et al, 1994; Tsukita et al, 1994; Nakanishi et al,
Immunostaining on cryostat and paraffin-embedded
sections of normal human skin (Fig 1) showed that the ep-
idermis was essentially negative for drebrin (Fig 1A). Drebrin
was, however, enriched in the ducts of eccrine sweat
glands, especially along their cell–cell boundaries (Fig 1B),
whereas sebaceous glands did not display detectable
amounts. In addition, intercellular junctions of hair follicles
were stained, both in the bulbs (Fig 1C) and, in upper parts,
in a layer apparently corresponding to the outer root sheath
(ORS; Fig 1D). As previously reported (Peitsch et al, 1999),
small blood vessels in the dermis were also drebrin-positive
(asterisks in Figs 1A and B). This specific distribution pattern
was observed in normal human skin from different parts of
the body such as the face, scalp, trunk, and foot sole, and
with all the various drebrin antibodies used.
1
997; Hirohashi and Kanai, 2003).
A protein recently identified at adhering junctions as well
as at lamellipodia and elongated cell processes of various
cell types is drebrin, an actin-binding protein (Peitsch et al,
1
999, 2001; Keon et al, 2000). This protein, originally de-
tected in neuronal cells (acronym for developmentally
regulated brain protein), can occur in four isoforms, drebrin
E1, E2, A, and sA (review: Shirao, 1995), only one of which,
E2, has so far been identified in non-neuronal cells. In neu-
ronal cells, the drebrins appear to be involved in the for-
mation of cell processes and in synaptic plasticity (e.g.,
Takahashi et al, 2003). Correspondingly, transfection of the
drebrin cDNA can cause formations of long actin-rich pro-
trusions and remodeling of the actin cytoskeleton (e.g.,
Shirao et al, 1992; Hayashi and Shirao, 1999; Keon et al,
To further specify the localization of drebrin within hair
follicles, double-label confocal microscopy was performed
on cryostat sections of scalp skin, using drebrin antibodies
in combination with marker proteins for the different hair
follicle compartments. Immunostaining of such sections for
drebrin and hair keratin 6hf, an intermediate filament protein
specific for the companion layer (Winter et al, 1998), showed
strong drebrin reactions in the companion layer. In the ORS,
2000), whereas antisense cDNA results in suppression of
neurite outgrowth (Toda et al, 1999).
In epithelial and endothelial cells, drebrin has been lo-
calized primarily to intercellular junctions (Peitsch et al,
00
drebrin labeling was somewhat weaker (Figs 2A–A , trans-
verse section; Fig 2B, longitudinal section) and depended
on the specific type of fixation (see Materials and Methods).
By contrast, when hair follicles were double labeled with
antibodies against drebrin and keratin 6irs1, a marker pro-
tein for the inner root sheath (IRS; Langbein et al, 2002), a
differential distribution of both proteins and absence of
drebrin from the IRS was noted (data not shown). The same
observation was made for drebrin antibodies in combination
with a marker specific for the Huxley layer of the IRS, hair
keratin 6irs4 (Fig 2C; Langbein et al, 2003b). Drebrin and
cytokeratin 14, one of the intermediate filament proteins
synthesized in the ORS, were found in the same layer but,
as expected, in a different localization: Drebrin was accu-
mulated along cell boundaries, whereas cytokeratin 14 was
positive throughout the cytoplasm (Fig 2D).
1
999), whereas in a series of other cell types, it appears to
be accumulated in actin filament-rich cell processes (Pei-
tsch et al, 2001, 2003). Drebrin has so far not been detected
in the skin and in skin tumors. Moreover, very little is known
about its distribution and function in stratified epithelia.
Therefore, we have generated a novel monoclonal drebrin
antibody that could be used, in combination with other an-
tibodies, to study the distribution of drebrin in normal skin,
epithelial skin tumors, and cultured keratinocytes. In addi-
tion, we have transfected cells of a related cell type, the
vulvar carcinoma line A431 devoid of endogenous drebrin,
with cDNA combining drebrin E2 with enhanced green flu-
orescent protein (EGFP) to allow the study of this protein by
immunocytochemistry and live cell microscopy.
Immunolocalization of drebrin in epithelial skin tumors
As the gene expression patterns of several junction-associ-
ated proteins are modified in skin tumors, our next aim was
to examine the distribution of drebrin in such tumors and
in precancerous lesions. Sections of frozen and paraffin-
embedded BCC, SCC, keratoacanthomas, and precursor
lesions such as actinic keratoses were systematically exam-
ined, using drebrin-specific antibodies (Table I). In BCC, in-
tense drebrin reactivity was observed in all specimens
examined (Fig 3A). This conspicuously positive reaction
was seen throughout the entire tumor on formaldehyde-fixed
cryostat sections and paraffin-embedded tissues. On cryost-
at sections fixed with acetone, the carcinoma cells located at
the periphery of the BCC sometimes appeared enhanced,
whereas reactions within the tumors were slightly weaker
(not shown).
Results
Drebrin distribution in normal human skin In previous
studies, we had noted drebrin staining along intercellular
junctions of certain epithelial and endothelial cell types (Pe-
itsch et al, 1999, 2003). But not much was known about
drebrin in stratified squamous epithelia, including the epi-
dermis, and tumors derived therefrom. Therefore, examina-
tion of the presence, localization, and dynamics of this
protein in human skin and related tissues as well as SCC
was overdue. As, in our former studies, the only available
monoclonal antibody (mab) to drebrin (clone M2F6) had
worked well on cultured cells but sometimes showed reac-
tions that were too faint or of dubious significance on tissue
sections, we decided to generate a novel mab against
drebrin that reliably reacted with all isoforms (mab Mx823;
see Materials and Methods). This antibody was used in par-
allel with the mab M2F6, a guinea-pig antiserum directed
against the same peptide (dreb4.1 gp) and two further
guinea-pig drebrin antisera (dreb254.2 gp and drebE2/A.2 gp).
In contrast to the strong and near-homogeneous drebrin
immunostaining of BCC, the drebrin distribution appeared to
be mostly inhomogeneous in SCC: usually, rather intense
drebrin reactions were noted along cell borders in certain
parts of the tumor, whereas other parts of the same tumor

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25 : 4 OCTOBER 2005
DREBRIN IN EPITHELIAL SKIN CANCERS 763
Figure 1
Immunolocalization of drebrin in paraf-
fin-embedded sections of normal hu-
man skin. Normal human epidermis
(bracket in A) contains almost no drebrin,
but immunoreactions are seen in the en-
dothelium of small blood vessels (aster-
isks in A and B). Intense labeling is noted
in the ducts of eccrine sweat glands (B),
especially along the cell-to-cell borders.
In addition, drebrin is enriched along the
intercellular junctions of hair follicles, both
in the bulb (C) and, in upper parts, in the
outer root sheath (D). Phase-contrast mi-
0
0
crographs are shown in A –D . A–D, anti-
serum dreb4.1gp. Scale bars: 25 mm.
appeared to be negative for drebrin (Fig 3B). The drebrin-
positive tumor regions were mostly found at the invasive
tumor front, but sometimes also occurred near horn pearls of
differentiated squamous carcinomas, in a distribution that
did not display a correlation to the specific degree of differ-
entiation. To examine whether the drebrin-rich cells within an
SCC represented the highly proliferative fraction, double
immunolabeling was performed with drebrin antibodies in
combination with others reactive with the proliferation marker
Ki67. Reactions for Ki67 were found in numerous cells at the
invasive tumor margins, but the Ki67-positive cells were not
identical to the drebrin-positive ones (data not shown).
The same phenomenon as in SCC, i.e. mosaicism of in-
tensely drebrin-positive and drebrin-negative tumor regions,
was observed in keratoacanthomas (Figs 3C, D). Precan-
cerous lesions such as actinic keratoses showed similar
heterogeneity. Here, in some regions, the basal keratin-
ocytes revealed some cell border drebrin staining, whereas
other basal regions and the upper epidermal layers re-
mained drebrin-negative (Table I).
Figure 2
Double-label confocal immunofluo-
rescence microscopy of human hair
follicles. Formaldehyde-fixed cryostat
sections of hair follicles were stained for
drebrin with monoclonal antibody Mx823
00
(red in A, A , and B–D), in combination
with antisera against different hair keratins
(
green). Both drebrin and hair keratin 6hf
0
00
(green in A , A , and B) are enriched in the
00
companion layer (A–A , vertical section;
B, longitudinal section). In addition, dreb-
rin antibodies react in the outer sheath
00
ORS) (A, A , and B–D) whereas the
(
Huxley layer of the inner root sheath,
labeled by antibodies against hair keratin
6
irs4 (green in C), does not present sig-
nificant amounts of drebrin (C, longitudi-
nal section). In D, co-existence of
cytokeratin 14 (green) and drebrin in the
00
ORS is shown. Asterisks in A and A de-
note drebrin-positive eccrine sweat gland
ducts. Scale bars: 100 mm.

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64 PEITSCH ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Table I. Drebrin distribution in epithelial skin tumors and precancerous lesions, compared with psoriasis and
seborrheic keratoses
Specimen
BCC
Number of samples
Drebrin distribution
13 (from 12 patients) Twelve of 13 (both nodular and sclerodermiforme BCC): Intense and
homogenous staining at cell borders throughout the tumor
Nodular
Sclerodermiforme
SCC
One nodular BCC (recurrence): intense and homogeneous staining in one area,
whereas other regions show only weak cell border staining
1
1
2
11 (from 10 patients)
Eleven of 11: significant cell border staining in certain cell groups, other cell
groups drebrin-negative
Well differentiated (G1)
G1 tumors: staining occasionally increased staining near horn pearls
6
4
Moderately differentiated (G2)
G2 and G3 tumors: in some tumors, staining at invasive margins; in average,
less drebrin-positive cell groups than in SCC of G1
Poorly differentiated (G3)
Keratoacanthomas
1
2
(G2–3)
Two of two: cell borders intensely drebrin-positive in certain regions; other areas
completely negative
Actinic keratoses
5
Five of five: some dysplastic keratinocytes in the basal epidermis drebrin-
positive; number of reactive cells and intensity variable
Psoriasis
9
8
Nine of 9: no drebrin reactions within the epidermis; small dermal blood
vessel show cell border staining
Psoriasis vulgaris
Psoriasis pustulosa palmoplantaris
1
4
Seborrheic keratoses
Four of four: drebrin-negative
BCC, basal cell carcinoma; SCC, squamous cell carcinoma.
To clarify whether drebrin synthesis might be correlated
with epidermal hyperproliferation in general, biopsy tissues
from patients with psoriasis were immunostained. In all of
the psoriasis samples examined, the epidermis was prac-
tically devoid of drebrin, indicating that drebrin most likely is
not related to hyperproliferation per se. Moreover, none of
the seborrheic keratoses examined revealed significant
amounts of drebrin (Table I).
We wondered whether the staining pattern observed
here was also applicable for other actin-binding proteins.
Therefore, we immunostained sections of human skin and
skin tumors with antibodies to ezrin, a protein sharing cer-
tain features with drebrin such as a prolin-rich, profilin-
binding domain and the tendency to accumulate in actin-
rich cell protrusions and in the cell cortex (for a review, see
Bretscher et al, 2002). In normal skin, ezrin was enriched at
the junctions of keratinocytes in all epidermal layers, in se-
baceous glands, eccrine sweat glands, and hair follicles,
and also at cell borders in endothelia of small blood vessels
such as a-catenin, b-catenin, and plakoglobin showed a far-
reaching overlap with drebrin (data not shown). In contrast,
the desmosomal plaque protein, desmoplakin, was com-
pletely differently distributed, showing the typical punctuate
desmosomal arrays whereas drebrin immunostaining dis-
played a more linear and, in some regions, distinctive in-
0
0
terdesmosomal pattern (Figs 4B–B ). When BCC were
labeled for proteins of tight junctions such as ZO-1, ZO-2,
and occludin, no significant reaction was noted. Claudin-1
was enriched along cell boundaries, but mostly differently
distributed from drebrin (not shown; for localization of clau-
din-1 in SCC, see Langbein et al, 2003a). With antibodies
against the gap junction protein connexin 43 faint, if any,
staining and no overlap with drebrin was seen in BCC (see
also Tada and Hashimoto, 1997), whereas keratinocytes
in the overlying epidermis displayed intense typical gap
junction-like reactions, serving as an internal control (not
shown). As other authors have recently observed co-local-
ization and biochemical interaction of drebrin and connexin
43 at gap junctions of astrocytes and Vero cells (Butkevich
et al, 2004), we double immunostained various cultured
cells, including primary human keratinocytes, mammary
carcinoma cells of line MCF-7, and endothelial human
umbilical vein endothelial cells for drebrin and connexin 43
but, again, mutually exclusive localization was noted.
(
data not shown). Immunostaining of different epithelial skin
tumors and precancers showed strong ezrin reactions at
cell boundaries and, although weaker, in the cytoplasm of
BCC, SCC, keratoacanthomas, and actinic keratoses, ho-
mogeneously throughout the lesions (not shown).
Double-immunofluorescence confocal laser scanning
and immunoelectron microscopy When cryostat sections
of BCC were double stained for drebrin and actin, almost
complete co-localization of both proteins along intercellular
borders was found (Figs 4A–A ). Double immunostaining
with different marker proteins of adhering junction plaques
As our double label laser scanning results indicated ac-
cumulation of drebrin near adhering junctions, we also
performed immunoelectron microscopy (Figs 5B–E). On
sections of BCC, immunogold labeling for drebrin was de-
tected in the microfilament network underlying the plaques
of adhering junctions, usually close to the plaques, whereas
00

1
25 : 4 OCTOBER 2005
DREBRIN IN EPITHELIAL SKIN CANCERS 765
Figure 3
Figure 4
Double-label immunofluorescence confocal microscopy, showing
drebrin accumulation at adhering junctions of basal cell carcinomas
Drebrin distribution in different epithelial skin tumors. Immuno-
histochemical reactions on sections through aldehyde-fixed, paraffin-
embedded tissues are shown. (A) In basal cell carcinoma (BCC), strongly
positive drebrin reactions throughout the tumor are observed, whereas
the overlying epidermis is drebrin-negative (brackets). In contrast, in
squamous cell carcinoma (SCC) (B) and in keratoacanthomas (C, D),
heterogeneous expression patterns are seen: certain groups of cells
exhibit marked drebrin enrichment at cell borders (B, C), whereas others
appear to be drebrin-negative (B, D). In C and D, two different regions
of the same keratoacanthoma, both containing horn pearls (HP), are
shown: although the HP-surrounding tissue in C is positive, that shown
in D is negative. The asterisks in D designate drebrin-positive blood
vessels, here also serving as an internal positive control. A–D, antiserum
(
BCC). When formaldehyde-fixed cryostat sections of BCC have been
0
double labeled for drebrin (mab Mx823, red in A and A ) and actin (phal-
00
loidin Alexa 488, green in A and A ), far-reaching co-localization of both
proteins in the submembranous actin cortex underlying adhering junc-
tions is seen (A, merge). In contrast, drebrin and desmoplakin antibodies
0 00
mab Mx823, red in B and B ; rabbit anti-desmoplakin, green in B and B )
(
display mutually exclusive localizations, as desmoplakin is restricted to
dot-like structures corresponding to desmosomes and drebrin occurs in
intermittent structures, representing an alternate pattern (B, merge). Note
also the occurrence of some cytoplasmic granular drebrin aggregates
(see also Peitsch et al, 1999, 2001). Scale bars: 10 mm.
0
0
dreb4.1gp; A –D , phase-contrast images. Scale bars: 25 mm.
our immunostaining results, and in SCC, despite the micro-
scopically observed heterogeneous staining pattern. In
contrast to drebrin, the amounts of actin were approx-
imately equal in all samples examined, with the expected
desmosomes were not significantly labeled. For compari-
son, immunoelectron microscopy with antibodies to b-cat-
enin was performed, which showed a typical junctional
plaque reaction, typically closer to the plasma membrane
than drebrin (data not shown).
00
exception of the actin-rich leiomyosarcoma (Fig 6A ).
Primary human keratinocytes synthesize drebrin When
primary human keratinocytes were analyzed by immunoflu-
orescence microscopy and immunoblotting (Fig 7), drebrin
could be detected in rather high amounts. Drebrin and actin
again co-localized along cell–cell boundaries, correspond-
ing to our observations on BCC (Fig 7A). Moreover, in sub-
confluent cultures, numerous actin-rich filopodia were
observed at the free margins of the cells but did not reveal
drebrin enrichment (data not shown). Immunoblot analysis
of total proteins from subconfluent (Fig 7B, lane 1) and
confluent (lane 2) keratinocytes showed strong drebrin re-
actions. Thus, the cell culture conditions appeared to have
Immunoblot analysis of skin and skin tumors To com-
pare the amounts of drebrin in the skin and in skin tumors,
we performed immunoblot analysis of total protein lysates
00
from normal skin (Figs 6A–A , lane 1), scalp (lane 2), BCC
lane 3), squamous carcinomas (lane 4), melanoma met-
(
astases (lane 5), and leiomyosarcoma (lane 6). An immuno-
reactive 120 kDa band corresponding to drebrin was hardly
detectable in normal skin and scalp but was drastically in-
creased in all tumors examined. Comparably high amounts
of drebrin were found in lysates of BCC, corresponding to

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66 PEITSCH ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 5
Electron and immunoelectron micros-
copy of basal cell carcinomas (BCC).
The conventional electron micrograph
presented in A shows two neighboring
BCC, connected by a region rich in junc-
tions, i.e., desmosomes (arrows) and pun-
ctum-like adhering junctions (asterisks),
the latter usually characterized by a small-
er junctional plaque. (B–E) When cryostat
sections of BCC have been reacted for
ultrathin section electron microscopy with
drebrin antibodies (B–E, monoclonal anti-
body Mx823), in combination with sec-
ondary gold-conjugated immunoglobulins
and silver enhancement, prominent reac-
tions are seen along intercellular adhering
junctions (B, C), sometimes close to, but
distinctly separate from desmosomal pla-
ques (D–E, higher magnifications). N, nu-
cleus. Scale bars: 500 nm (A, B); 200 nm
(
C–E).
induced drebrin synthesis in normally near drebrin-negative
keratinocytes.
drebrin–EGFP–N1 transfectants (lane 6), two prominent
bands were seen: one at approximately 140 kDa in the
EGFP–C1 lines or 150 kDa in the EGFP–N1 lines, and the
other at approximately 43 kDa. These bands were identified
as drebrin and actin by ‘‘peptide finger printing,’’ indicative
of an actin–drebrin complex (for similar complexes, see also
Hayashi et al, 1999). Four other minor bands were also ex-
cised and analyzed by trypsin digestion and matrix-assisted
laser desorbtion/ionization (MALDI) and were identified as
different myosin isoforms. Compared with drebrin and actin,
these bands were much fainter and also appeared, in trace
amounts, in control samples. Similar immunoprecipitation
results were obtained with antisera dreb254.2 (Fig 8B) and
mab Mx823 (data not shown). As profilin, another actin-
binding protein interacting with prolin-rich motifs, has been
described as a binding partner for drebrin (Mammoto et al,
1998), our immunoprecipitates were also immunoblotted for
profilin, however, without significant reactions (data not
shown).
Generation and analysis of drebrin cDNA-transfected
cell lines Currently, the regulation of the formation of actin-
rich cell processes is discussed as a major function of
drebrin (e.g., Shirao, 1995), whereas not much is known
about its functions in relation to adhering junctions. To elu-
cidate such functions, we generated cell lines stably ex-
pressing drebrin–EGFP fusion proteins. We chose A431
cells as this was one of the rare cell lines devoid of endog-
0
enous drebrin (Fig 8A , lane 2; see also Peitsch et al, 1999,
2
001). Such cells were transfected—transiently and sta-
bly—with constructs of drebrin and EGFP (both C- and N-
terminal) and with myc-tagged drebrin, with essentially
identical results. With all constructs used, some of the
transfectants overexpressed drebrin, usually displaying nu-
merous actin-rich cell processes (data not shown; for rel-
evant references, see Introduction). In the transfectants
seemingly containing less drebrin, however, the fusion pro-
tein was enriched at cell–cell boundaries, and fewer and
shorter cell processes were seen (Fig 9, left panel). For fur-
ther experiments, stable lines exhibiting the latter pheno-
When A431 cells stably transfected with drebrin–EGFP
constructs were immunostained with antibodies and com-
pared with constituent proteins of intercellular junctions,
far-reaching co-localization was evident (Fig 9; Table S1).
0
0
00
type were analyzed. Three such lines (Fig 8A , lanes 3–5)
Antibodies against plakoglobin (Figs 9A , A ), a protein of
the plaques of both adhering junctions and desmosomes
(Cowin et al, 1986), revealed some co-distribution with the
drebrin–EGFP fusion proteins but the typical punctate des-
mosomal structures were drebrin negative. Proteins repre-
senting exclusive ‘‘markers’’ for adhering junctions such
were examined in comparison with non-transfected A431
cells (lane 2) and human astrocytic glioma cells (lane 1) and
were found enriched in drebrin, in levels comparable with
those in glioma cells.
To test whether the drebrin–EGFP fusion proteins also
interacted with actin, as shown for the endogenous protein,
lysates from stable transfectants were immunoprecipitated
with drebrin antibodies (Fig 8B). In immunoprecipitates
of both drebrin–EGFP–C1 transfectants (lane 4) and of
0
00
as the plaque proteins b-catenin (Figs 9B , B ), a-catenin,
and p120 (not shown) as well as the transmembrane protein
0
00
E-cadherin (Figs 9C , C ) partially co-localized with drebrin
00
0
(see merged images in Figs 9B , B ). When cells were

1
25 : 4 OCTOBER 2005
DREBRIN IN EPITHELIAL SKIN CANCERS 767
known to occur both at adhering and at tight junctions.
Other tight junction proteins such as occludin, claudin 1,
and ZO-2 were not detected in monolayer cultures of the
transfectants. Similarly, no significant reaction was seen
for the gap junction protein connexin 43 (not shown). An-
0
tibodies against a-actinin (not shown), vinculin (Figs 9D ,
0
0
D ), and vasodilator-stimulated phosphoprotein (VASP) (not
shown) reacted predominantly with focal adhesions that
were virtually devoid of drebrin–EGFP, as previously report-
ed for endogenous drebrin (Peitsch et al, 1999).
Taken together, the drebrin–EGFP fusion protein was
seen at adhering junctions, together with actin, close to, but
mostly not directly at the adherens plaques, corresponding
to our observations in situ.
Distribution of drebrin fusion proteins after stimulation
with epidermal growth factor (EGF) As drebrin is thought
to function as a regulator of cell processes in neurons (e.g.,
Toda et al, 1999), we examined whether this protein also
accumulates in or at newly forming protrusions. To this end,
we took advantage of the fact that A431 cells can rapidly
form lamellipodia and filopodia on stimulation with EGF (cf.,
e.g., Malliri et al, 1998). When stably drebrin-transfected
A431 cells were treated with 100 ng per mL EGF and analy-
zed by live cell microscopy, rapid formation of lamellipodia
was observed within a few minutes (Figs 10B, C; Supple-
mentary Material, Movie 1). Within the next minutes, the
lamellipodia retracted (Figs 10D, E), and filopodia-like
structures appeared (Figs 10F–H). During this process, the
drebrin–EGFP fusion proteins remained for the most part
0
0
enriched along cell boundaries (Figs 10A –H ; Supplemen-
tary Material, Movie 2), and after retraction even showed
0
enhanced fluorescence at these sites (arrows in Figs 10G ,
0
H ). In some cells, drebrin–EGFP was, in addition, tempo-
0
rarily enriched in spikes in extended (asterisks in Figs 10B ,
0
0
0
C ) and retracting lamellipodia (asterisks in Figs 10D , F ).
This signal, however, was rather faint compared with the
junctional signal, and the filopodia formed after approxi-
mately 15 min were virtually free of drebrin. Essentially, the
same observations were made when cells treated with EGF
for 5–15 min were fixed with formaldehyde, when cells were
stimulated with different EGF concentrations, ranging from
10 to 100 ng per mL (not shown), and with EGFP constructs
at the N- and the C-terminus of drebrin.
Figure 6
Increased amounts of drebrin in different skin tumors, as shown by
immunoblotting. Total proteins of normal human skin (lane 1), human
scalp (lane 2), a basal cell carcinoma (lane 3), a squamous cell carcinoma
(lane 4), a subcutaneous metastasis of a melanoma (lane 5), and a le-
iomyosarcoma (lane 6), have been loaded in approximately equal
amounts and separated by sodium dodecylsulfate-polyacrylamide gel
electrophoresis (A, Coomassie Brilliant Blue-stained gel). Immunoblot
In summary, our observations that drebrin is not accu-
mulated in the filopodia of subconfluent keratinocytes and
the live cell microscopy results all indicate that in the ep-
ithelial cells examined, drebrin normally functions at junc-
tions, not in filopodia and lamellipodia. Overexpression of
drebrin, however, may lead to the formation of cell proc-
esses, as previously described for other cells (e.g., Shirao
et al, 1992; Hayashi and Shirao, 1999; Keon et al, 2000).
0
analysis, using antibodies to drebrin (A ), shows a very faint immuno-
reactive 120 kDa band in normal skin and scalp, which is drastically
increased in all tumors examined. That lane 2 does not show a much
stronger signal than lane 1 may be because of the excess of inter-
00
follicular skin. (A ) In contrast to the drebrin band, the 43 kDa band of
actin appears at approximately equal intensities in all samples, with the
exception of the leiomyosarcoma containing a very large amount of actin
(lane 6). Molecular weight standards are as indicated on the left margin.
labeled for l/s-afadin, however, a junction plaque protein
forming complexes with the transmembrane proteins nectin
and ponsin (Takai and Nakanishi, 2003), little if any co-
distribution with drebrin–EGFP was seen (not shown).
Antibodies against desmosomal proteins such as des-
moplakin showed punctate staining patterns entirely differ-
ent from those of the drebrin fusion proteins (not shown).
No significant co-localization was seen with protein ZO-1,
Discussion
Drebrin in normal and pathologically altered tissues In
normal human skin, the epidermis contains very little, if any,
drebrin, but this protein is enriched in the ducts of eccrine
sweat glands, similar to what has been noted in mammary
gland ducts and in distal renal tubules (Peitsch et al, 1999,

7
68 PEITSCH ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 7
Induction and localization of drebrin in
primary cultured keratinocytes. When
human keratinocytes cultured for two
passages have been stained with drebrin
0
antibodies (red in A and A , monoclonal
antibody Mx823), in combination with
phalloidin to visualize actin filaments
00
(
green in A and A ), confocal laser scan-
ning microscopy shows accumulation of
drebrin along cell–cell borders, suggestive
of a localization at intercellular adherent
0
junctions (A ), in far-reaching co-localiza-
0
0
tion with phalloidin (A ). By contrast, the
cytoplasmic actin stress fibers do not
contain drebrin (A, merge). Scale bar: 10
0
mm. (B, B ) Using sodium dodecylsulfate-
polyacrylamide gel electrophoresis and
immunoblotting, a strongly reactive 120
kDa band corresponding to drebrin can
be detected in whole-cell lysates of both
subconfluent (lane 1) and confluent (lane
2
) keratinocyte cultures, in approximately
0
equal amounts (B ; B, Coomassie Blue-
stained gel). Molecular weight standards
in lane M are, from top to bottom, 212,
158, 116, 97, and 66 kDa.
2
003; Keon et al, 2000). Moreover, drebrin is accumulated in
enin and plakoglobin have been shown to control hair follicle
morphogenesis and cycling (Charpentier et al, 2000; Vasiou-
khin et al, 2001), and mutations and deletions in the
plakoglobin gene are associated with hair abnormalities
(‘‘Naxos disease’’; McKoy et al, 2000). To elucidate whether
drebrin might similarly be involved in follicular morphogen-
esis, we will study hair follicle development, murine hair fol-
licle cycling, and different types of alopecias.
The most remarkable result of our study is the strong
increase of drebrin in epithelial skin tumors. In particular,
intense and homogeneous immunostaining has been noted
in BCC, an intriguing finding in the light of the hypothesis of
a follicular derivation of BCC (e.g., Schirren et al, 1997; Jih
the ORS and the companion layer of hair follicles, most likely
in association with intercellular adhering junctions (zonulae
adhaerentes). This distribution pattern appears rather unex-
pected, as most proteins associated with adhering junctions
in hair follicles also occur in one or more epidermal layers
and vice versa. Several other junction-associated proteins,
however, are known to be specifically synthesized during hair
follicle development (review: Jamora and Fuchs, 2002).
Moreover, b-catenin (Gat et al, 1998; Celso et al, 2004) and
other plaque proteins of adhering junctions such as a-cat-
Figure 8
Immunoblot analysis and immunoprecipitation of drebrin-contain-
ing proteins in A431 cells stably transfected with drebrin-enhanced
0
green fluorescent protein (EGFP) constructs. (A, A ) Total proteins of
cultured astrocytic glioma cells (lane 1, positive control), non-transfected
A431 cells (lane 2), two different A431 lines stably transfected with
drebrin–EGFP–C1 constructs (lanes 3 and 4), and one of the stable
drebrin-EGFP–N1 transfectants (lane 5) have been gel electrophoretically
separated and either stained with Coomassie Blue (A) or immunoblotted
0
with drebrin antibodies (A , mab Mx823). Although non-transfected A431
cells do not contain detectable amounts of drebrin, intense reactions are
noted in the drebrin–EGFP–C1 transfectants at approximately 140 kDa
and in the drebrin–EGFP–N1 transfectant at approximately 150 kDa. The
slightly different gel electrophoretic mobilities of the constructs with
drebrin at the C- (lanes 3 and 4) and N-terminus (lane 5) of EGFP are
probably a result of a residual conformational difference under these
conditions. (B) When A431 cells stably transfected with drebrin–EGFP–
C1 (lane 4) and –N1 constructs (lane 6) are immunoprecipitated with
drebrin antibodies (antiserum dreb254.2gp), two major bands appear in
the Coomassie-stained precipitates: one at 140 kDa (lane 4, asterisk) or
approximately 150 kDa (lane 6, asterisk), identified as drebrin by peptide
finger printing, and one at approximately 43 kDa (lanes 4 and 6, arrows),
representing actin. In non-transfected A431 cells, used for immunopre-
cipitation control (lane 2), the only prominent band is seen at approx-
imately 55 kDa, containing the heavy chains of the immunoglobulins (see
also lanes 4 and 6). The proteins bound non-specifically during ‘‘pre-
clearing’’ of each lysate have been loaded for control (lanes 1, 3, and 5).
Protein standards in lane M (A, B) correspond to 212, 158, 116, 97, 66,
55, and 43 kDa (from top to bottom).

1
25 : 4 OCTOBER 2005
DREBRIN IN EPITHELIAL SKIN CANCERS 769
Figure 9
Drebrin–enhanced green fluorescent
protein (EGFP) fusion proteins localize
to adhering junctions. A431 cells stably
transfected with drebin–EGFP–C1 (green
in A–D) were immunostained with anti-
bodies to different junctional proteins (red
0
0
in A –D ) and analyzed by confocal laser
scanning microscopy. In the left panel,
showing localization of the drebrin–EGFP
constructs (A–D), the drebrin reaction is
predominantly observed along intercellu-
lar borders, although some diffuse cyto-
plasmic staining is also seen. Additionally,
some cells form numerous small cell
processes, extending to different focal
0
0
00
planes (asterisks in A, A , D, and D ).
When simultaneous staining is performed
with antibodies to the adhering junction-
0
associated proteins plakoglobin (A ), b-
catenin (B ), and E-cadherin (C ), the
0
0
00
00
merge images (A –C ) show partial co-lo-
calization with drebrin–EGFP at cell–cell
borders, more prominently for b-catenin
and E-cadherin than for plakoglobin.
Vinculin antibodies react with focal con-
tacts (D ), that do not contain drebrin (D ,
merge). Scale bars: 20 mm.
0
00
et al, 1999; Kr u¨ ger et al, 1999). Moreover, cytokeratin 6hf, a
keratin specific for the companion layer, has been detected
in more than 70% of the BCC examined (Kurzen et al, 2001),
in the absence of all other hair keratins (Cribier et al, 2001).
Interestingly, in the hair follicle, this layer is also conspic-
uous by its drebrin content. A follicular derivation of BCC
might explain why BCC are homogeneously drebrin posi-
tive, in contrast to the other epithelial skin tumors examined.
Whether the demonstrated reliable detection of drebrin in
BCC will be of practical diagnostic use has to be seen.
Reasonable diagnostic applications would be the possibility
of the detection of early BCC that might otherwise be over-
looked and of small nests at the margins of surgical resec-
tions. Moreover, it will also have to be examined as to
whether high amounts of drebrin occur in other tumors de-
rived from hair follicles such as pilomatrixomas, follicular
harmatomas, and pilomatrix carcinomas.
In contrast to BCC, SCC and their precursor lesions
as well as keratoacanthomas show surprisingly variable
immunoreactions for drebrin and immunostaining mosaic-
ism in the same tumor, without obvious relation to the local
degree of differentiation or proliferation. Mosaic patterns
within a tumor have also been found for a number of other
junctional proteins such as plakophilin 1 (Moll et al, 1997)
and desmosomal cadherins (Kurzen et al, 2003). In a recent
study, Langbein et al (2003a) have described tight junction
marker proteins defining subregions in SCC. Such data in-
dicate that epithelial skin cancers can comprise multiple
small subregions and that such regionalization might con-
tribute to notorious differences of chemoresistency of sev-
eral SCC.
In general, however, our western blot analyses have
shown highly significant drebrin increases in SCC, in levels
comparable with those in BCC. Moreover, drebrin has been

7
70 PEITSCH ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 10
Live cell microscopy of drebrin–enhanced green fluorescent protein (EGFP) transfectants after stimulation with epidermal growth factor
EGF). When drebrin–cDNA-transfected A431 cells are analyzed by live cell microscopy immediately after the addition of EGF, the bright images
show rapid formation of lamellipodia (A–C) that are most extended after approximately 4 min (C). In the following minutes, retraction of lamellipodia
D, E) and formation of numerous filopodia-like cell processes (F–H) are observed. During this process, most of the drebrin–EGFP fusion protein
(
(
0
0
0
0
retains a junctional localization (A –H ). In some cells, the junctional EGFP signal first seems to decrease slightly (C –E , cells in the lower left) but
0
0
then, after 20 min, appears to enhance again, in parallel with cell retraction (arrows in G and H ). Some minor amounts of the fusion protein are
0
0
additionally seen within protruding and retracting lamellipodia, here in streaky structures presumably representing actin spikes (asterisks in B –F ),
whereas no significant signals are seen in filopodia. Scale bar: 10 mm.
found to be markedly increased in melanoma metastases
and in leiomyosarcomas. This raises the question as to
whether upregulation of drebrin might be an event com-
monly related to tumorigenesis, which will have to be sys-
tematically examined in future studies. In this context, our
finding that drebrin is virtually undetectable in biopsy spec-
imens of patients with psoriasis, a hyperproliferative but not
tumorigenic skin disorder, might also be considered as a
hint in this direction. As to other proteins associated with
adhering junctions, b-catenin is widely regarded as a reg-
ulator of tumorigenesis (review: Nelson and Nusse, 2004;
see also Chan et al, 1999; Doglioni et al, 2003; El-Bahrawy
et al, 2003).
er content of drebrin (Fig 7) may reflect an actual transient
upregulation because of the adaption to culture conditions.
From this study and previous studies on epithelial cells, a
good portion of the cellular drebrin appears to enrich at
the cytoplasmic side of adhering junctions, in keratinocytes
particularly at the numerous small puncta adhaerentia
known to harbor a- and b-catenin and to anchor the actin
microfilament bundle system (see, e.g., Green et al, 1987;
Kaiser et al, 1993; K u¨ bler and Watt, 1993; Haftek et al,
1996). Similarly, in our drebrin cDNA transfections of A341
cells, a squamous carcinoma line devoid of endogenous
drebrin, the resulting fusion proteins are enriched at adher-
ing junctions and largely remain in this association after
stimulation with EGF. By contrast, in cells with an increased
drebrin content, such as in transfectants overexpressing the
cDNA constructs, an increased portion of drebrin appears
away from cell–cell boundaries. In such cells, we have
observed the formation of long, neurite-like cell processes,
in correspondence with results of other groups in other cell
Drebrin localization and morphogenic effects in epithe-
lial cells Previously, we have reported (Peitsch et al, 1999)
that permanently growing, but non-malignant keratinocytes
such as those of line HaCaT contain very little drebrin. That
primary keratinocytes (passage 2) show an apparently high-

1
25 : 4 OCTOBER 2005
DREBRIN IN EPITHELIAL SKIN CANCERS 771
types (Shirao et al, 1992; Hayashi and Shirao, 1999; Toda
et al, 1999).
tag with Eco RI and Xho I restriction. All PCR products were ver-
ified by DNA sequencing.
Whereas in neuronal cells drebrin is thought to function
primarily as a regulator of cell processes, the distribution
observed in epithelial cells implicates that here the protein
might be involved in the regulation of actin filament attach-
ment to adhering junctions. Moreover, drebrin has also been
implicated in cell–substratum adhesions in neuronal (Ikeda
et al, 1995) and in endothelial cells (our unpublished results).
There is increasing evidence for regulatory interconnections
between these pathways (e.g., Kovacs et al, 2002; DeMali
and Burridge, 2003). Taken together with our previous ob-
servations and with reports from the literature, the results
shown here suggest that drebrin can serve diverse impor-
tant functions in different cell types, including formation of
cell processes, cell–cell, and cell–substratum adhesion and
cell migration. Future studies will have to show whether and
in what way drebrin also contributes to the special cell mi-
gratory and adhesive activities of tumor cells.
Transient and stable transfection of A431 cells Cultured A431
vulvar carcinoma cells were transiently transfected with drebrin–
pEGFP–C1, –N1, and myc-tagged constructs at cell densities of
70%–90%, using lipofectamine 2000 (Invitrogen) according to the
manufacturer’s instructions. To generate stable lines, A431 cells
transfected with drebrin–pEGFP–C1 and –N1 were seeded on 10
cm-culture dishes at a 1:100 dilution and selected with 1.2 mg per
mL geniticin (Invitrogen). Geneticin-resistant colonies were picked
with 3 mm-cloning discs (Sigma) and transferred first into 24-well
microtiter plates, and then into 2 cm-culture dishes. Drebrin-pos-
itive clones were selected according to EGFP fluorescence and by
immunoblot analysis of total protein lysates. Stably transfected
A431 lines were maintained in DMEM supplemented with 10% fetal
calf serum, 2 mM glutamine, and 1 mg per mL geniticin.
Tissues Samples of human skin and skin tumors were obtained in
the course of routine pathological diagnoses from the Departments
of Dermatology and Pathology of the Medical Center Mannheim.
For some experiments, samples were snap-frozen in liquid nitro-
gen at approximately ꢀ1301C and stored at ꢀ801C. For other ex-
periments, aldehyde-fixed, paraffin-embedded tissues were used.
Procedures were performed with patients’ informed consent.
Materials and Methods
Cell cultures Human vulvar squamous carcinoma cells of line
A431 American Type Culture Collection (Manassas, Virginia) were
grown in Dulbecco’s Minimal Essential Medium (DMEM; Invitrogen,
Karlsruhe, Germany) supplemented with 10% fetal calf serum
Generation of monoclonal and polyclonal antibodies to
drebrin For the generation of mab to drebrin, we used a cocktail
of three different peptides derived from the amino acid sequence,
pep1 (amino acids (aa) 32–44, YTYEDGSDDLKLA), pep3 (aa
(
Biochrom, Berlin, Germany) plus 2 mM glutamine, and were sub-
487–502, STLQGEPRAPTPPSGT), and pep4 (aa 632–649,
divided twice weekly at a 1:10 ratio. For stimulation with EGF (Si-
gma, Deisenhofen, Germany), cells were fed with serum-free
medium and, after 2 d, EGF was added in concentrations between
CWDADPVPEEEEGFEGGD), all of which were synthesized by
Peptide Specialty Laboratories (Heidelberg, Germany). Three 9-
wk-old female BALB/c mice were immunized by subcutaneous
injections of 100 mg of the peptide cocktail diluted in phosphate-
buffered saline (PBS) to a volume of 150 mL, supplemented with
1
0 and 100 ng per mL. Stimulated cells and non-stimulated con-
trols were further analyzed 0–25 min after stimulation. Primary hu-
man keratinocytes were grown using Keratinocyte Growth Medium
150 mL of complete Freud’s adjuvants (Sigma). Three subcutane-
2
Kit (PromoCell, Heidelberg, Germany) at normal calcium con-
ous booster injections with the same amount of peptide cocktail
mixed with 150 mL incomplete Freud’s adjuvant were given at in-
tervals of 21 d. At day 63, a final boost with 30 mg of the peptide
cocktail diluted in 200 mL PBS was injected intraperitoneally. Four
days thereafter, the mice were sacrificed, spleen cells were har-
vested and fused with mouse myeloma cells of line SP2/0 at a ratio
of 2:1 in the presence of polyethylenglycol 1500 (Roche Diagnos-
tics, Mannheim, Germany). After fusion, the cells were placed on
24-well microtiter plates and cultured in hypoxanthine–amino-
pterin–thymidine (HAT)-containing RMPI-1640 medium (Invitrogen)
supplemented with 10% fetal calf serum (Invitrogen) and 10%
Condimed H1 (Roche Diagnostics). Hybridomas were screened by
immunofluorescence microscopy on cultured glioma cells (cf. Pei-
tsch et al, 2001) and immunoblot reactivity on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated
lysates of glioma cells. Positive colonies were subcloned twice by
limited dilution in 96-well microtiter plates. After subcloning, hy-
bridomas were weaned off HAT–RMPI into RMPI medium alone
centration (1.5 mM CaCl ). Cells were subcultured once per week
2
at a 1:3 ratio, and experiments were conducted mostly after the
second passage. The medical ethical committee of the Medical
Center Mannheim, University of Heidelberg, approved all de-
scribed studies. The study was conducted according to the Dec-
laration of Helsinki Principles.
Cloning of drebrin–EGFP constructs and myc-tagged con-
structs The full-length cDNA encoding drebrin E2 (IMAGE ID:
3
(
356428) was obtained from Resource Center/Primary Database
RZPD, clone ID: IMAGp958A05162, in a pOTB7 vector). For clon-
ing into vector pEGFP–C1 (BD Biosciences Clontech, Palo Alto,
California), a Bgl II restriction site was generated by PCR at the N-
0
terminus of the drebrin E2 sequence (forward primer: 5 -TTT AGA
0
0
TCT GCC GGC GTC AGC TTC AGC GGC-3 ; reverse primer: 5 -
CGC ACT TGC GGG CAT CAG). The drebin cDNA was cloned into
the Bgl II and Bln I sites of the pEGFP–C1 vector.
For cloning into vector pEGFP–N1 (BD Biosciences Clontech), a
Bam H1 restriction site at the C-terminus of the drebrin sequence
was created by PCR using a reverse primer containing both an Xho
2
and grown in 25 cm flasks or propagated as ascites in Pristane-
treated BALB/c mice. Immunoglobulin subclasses were deter-
mined and one of the drebrin-specific immunoglobulin G (IgG) mab
obtained, Mx823, was further characterized by separate preincu-
bation with the peptides used for immunization at concentrations
ranging from 10 to 200 mg per mL in PBS, followed by immuno-
fluorescence microscopy and immunoblot analysis. These exper-
iments provided evidence that mab Mx823 was directed against
pep4, located at the drebrin C-terminus.
Mab Mx823 can be applied in immunolabeling of methanol-,
acetone-, and formaldehyde-fixed cells and tissues, showing the
strongest reactions after formaldehyde fixation (optimal dilution:
1:50), but not on paraffin-embedded tissues. The antibody also
efficiently immunoprecipitates drebrin and is now available from
Progen Biotechnik (Heidelberg, Germany).
0
I and a Bam HI site (forward primer: 5 -GGT ACT TCA GTC AAT
0
0
CAC AGG-3 ; reverse primer: 5 -TTT CTC GAG GAT CCT CAC CAC
0
CCT CGA AGC CCT CCT C-3 ), followed by Sac I/Xho I disgestion.
Cloning into the pEGFP–N1 vector was performed using Eco RI
and Bam H1 sites.
To generate a myc-tagged drebrin construct, a purified, Hind III/
Eco R1 digested myc-tag was cloned into the HindIII and EcoR1
sites of vector pcDNA3.1 (Invitrogen). An EcoR1 restriction site at
the N-terminus of the drebrin sequence was generated by PCR on
0
the drebrin–pOTB vector (forward primer: 5 -AAG AAT TCA ATG
0
0
GCC GGC GTC AGC TTC AGC-3 ; reverse primer: 5 -CGC ACT
0
TGC GGG CAT CAG GCA CAT-3 ). The drebrin sequence was
subsequently cloned into the pcDNA3.1 vector containing the myc-

7
72 PEITSCH ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Pep4 was also used for generation of antibodies in guinea-pigs
cf. Peitsch et al, 2001). The resulting antiserum dreb4.1gp also
Electron and immunoelectron microscopy Conventional elec-
tron microscopic analysis of samples of BCC was performed ac-
cording to the protocol described in Peitsch et al (2003). For
immunoelectron microscopy, cryostat sections of BCC of 4–5 mm
thickness were fixed with 2% formaldehyde for 10 min (RT), fol-
(
reacted well on aldehyde-fixed, paraffin-embedded sections after
antigen retrieval. Other drebrin-specific guinea-pig antisera used,
i.e., dreb254.2 and drebE2/A.2, the latter being specific for the
isoforms drebrin E2 and A, have been previously described (Pe-
itsch et al, 2001, 2003).
4
lowed by treatment with 50 mM NH Cl (5 min) and permeabilization
with 0.1% saponin (5 min). Staining and embedding were as pre-
viously reported (Peitsch et al, 2003). Primary antibodies were
Mx823 (1:50) and the rabbit antiserum to b-catenin, secondary
antibodies anti-mouse, or anti-rabbit immunoglobulins conjugated
with 1.4 nm-gold particles (Nanogold; Biotrend, K o¨ ln, Germany).
Silver enhancement was performed for 5 min with a silver en-
hancement kit from Nanoprobes (Stony Brooks, New York, New
York). Electron micrographs were taken with an EM 900 elecron
microscope (LEO, Oberkochen, Germany) at 80 kV.
Other antibodies and reagents For comparison, we also used the
murine drebrin mab M2F6 against purified chicken drebrin E, pur-
chased from MoBiTec (G o¨ ttingen, Germany). Further mab applied
in this study were against plakoglobin (clone PG5.1, Progen Bio-
technik, and clone 11E4, kindly provided by Dr M. J. Wheelock,
University of Nebraska Medical Center, Omaha, Nebraska), des-
mogleins Dsg 1 and 2 (clone DG3.10; Progen Biotechnik), E-cadherin,
ctn
proteins p120 , VASP, and Mena (all from BD Biosciences Phar-
mingen, Heidelberg, Germany), vinculin (clones 11.5 and hvinc-1),
Immunoblotting, immunoprecipitation, and MALDI analy-
sis Immunoblot analysis of lysates of cultured cells and tissues
was performed as previously described (Peitsch et al, 1999). For
immunoprecipitation, cultured cells were washed twice with pre-
cooled PBS, lysed in IP buffer (0.1% NP-40, 150 mM sodium
chloride, and 20 mM N-2-hydroxyethylpoperazine-N-2-ethane-
sulfonic acid, pH 7.4, supplemented with Complete Mini inhibitor
tabs, EDTA free, from Roche Diagnostics) for 1 h on ice and were
mechanically homogenized. After centrifugation for 10 min at 14,000
rpm (41C), the supernatants were ‘‘precleared’’ by incubation
with protein A-coupled dynabeads or pan-mouse-IgG-coupled
dynabeads (Dynal Biotech, Hamburg, Germany) for 2–3 h (41C).
Immunoprecipitation of drebrin was performed overnight, using
either protein A-dynabeads preincubated with the drebrin antisera
dreb254.2 or drebE2/A.2 diluted in a 50 mM TRIS buffer (pH 7.5) or
pan-mouse-IgG-dynabeads preincubated with mab Mx823 in
TRIS buffer. The immunoprecipitates were washed five times
in IP-buffer, solubilized in thrice concentrated Laemmli sample buff-
er (Peitsch et al, 1999), boiled for 3 min at 951C, and subjected to
SDS-PAGE. The precipitates obtained after preclearing, containing
the non-specifically bound proteins, were treated identically
and used as controls. Some of the electrophoretically separated
probes were transferred onto polyvinylidene difluoride membranes
and analyzed by immunoblotting. Others were stained with
Coomassie Brilliant Blue; the bands were excised and processed
for ‘‘peptide finger printing.’’ Trypsin digestion and MALDI analysis
were performed in cooperation with Dr M. Schn o¨ lzer (Protein
Analysis Facility, German Cancer Research Center), as described
ezrin and a-actinin (all from Sigma), actin (clone 2G2), cytokeratin
18 (clone 174.1), and profilin (all from Progen Biotechnik), and Ki67
as proliferation marker (Dianova, Hamburg, Germany).
Rabbit antisera against a-catenin, b-catenin, and l/s-afadin
were obtained from Sigma, rabbit antibodies against proteins ZO-
1
, ZO-2, occludin, claudin-1, and connexin 43 from Zymed Lab-
oratories (San Francisco, California), and rabbit antibodies against
desmoplakin from NatuTec (Frankfurt/Main, Germany). Guinea-pig
antisera against cytokeratin 14 or the hair keratins 6hf, 6irs1, and
6
irs4 were kindly provided by Dr Lutz Langbein (Division of Cell
Biology, German Cancer Research Center, Heidelberg, Germany;
cf. Winter et al, 1998; Langbein et al, 2002, 2003b). Actin micro-
filaments were stained with phalloidin coupled to Alexa 488 (Mo-
BiTec) or TRITC-labeled phalloidin (Sigma).
For immunofluorescence microscopy, primary antibody com-
plexes were visualized with secondary antibodies coupled to Cy-3
(
Dianova) or Alexa 488 (MoBiTec). For immunoblot analysis, horse-
radish peroxidase-conjugated secondary antibodies were applied
in combination with the enhanced chemiluminescence system
(
NEN, K o¨ ln, Germany).
Immunofluorescence and confocal laser scanning micros-
copy Cultured cells grown on glass coverslips were fixed in 2%
formaldehyde for 20 min at room temperature (RT) and treated with
5
4
0 mM NH Cl for blocking of free aldehyde groups (5 min, RT),
followed by one wash in PBS and by permeabilization in 0.1%
Triton X-100 for 3 min (RT). Alternatively, fixation was performed
with methanol (ꢀ201C, 5 min) and acetone (ꢀ201C, 20 s). Frozen
tissues were sectioned at 4–5 mm thickness, using a Leica cryo-
microtome (Bensheim, Germany), air-dried and fixed with 2% for-
maldehyde or, alternatively, with acetone (ꢀ201C, 10 min). Prior to
immunostaining, sections were blocked with 5% goat serum for 20
min. For sectioning of paraffin-embedded tissues (3–4 mm), a Jung
Histoslide 2000R cryomicrotome (Leica) was used. After de-
paraffinization according to standard techniques, sections were
pre-treated by microwaving in 100 mM TRIS buffer containing 5%
urea (pH 9.5, 10 min, 981C) to achieve heat-induced antigen re-
trieval. Sections were then washed with PBS (5 min, RT), incubated
with PBS containing 2% milk powder (Carl Roth, Karlsruhe, Ger-
many) for 10 min, and blocked with 10% goat serum and 2% milk
powder in PBS (15 min). Immunostaining, immunofluorescence
microscopy, and confocal laser scanning microscopy were per-
formed as described (Peitsch et al, 2001), using an Axiophote II
photomicroscope (Carl Zeiss, Jena, Germany) for immunofluores-
cence microscopy and a Zeiss LSM 510 UV microscope for con-
focal microscopy.
(Peitsch et al, 1999).
The authors thank Dr Lutz Langbein (Division of Cell Biology, German
Cancer Research Center) for providing antibodies specific for hair
keratins and for helpful discussion, as well as Prof. Eberhard Spiess
(
Biomedical Structure Analysis Group, German Cancer Research Cen-
ter) for advice on live cell microscopy. Dr Martina Schn o¨ lzer (Central
Protein Analysis, German Cancer Research Center) kindly performed
MALDI analyses. Silke Pr a¨ tzel (Division of Cell Biology, German Cancer
Research Center) is acknowledged for technical support in electron
microscopy, Ralf Zimbelmann (Division of Cell Biology, German Cancer
Research Center) for support in cloning, and Christel Herbst (Depart-
ment of Dermatology, Medical Center Mannheim) for keratinocyte cell
culture. In addition, we would like to thank Dr Jochen Utikal (Depart-
ment of Dermatology, Medical Center Mannheim) for help with pres-
ervation of tumor material and Dr Romy Porstmann (Department of
Pathology, Medical Center Mannheim) for aid in pathological diagnos-
tics. This work was supported by a grant from the Deutsche Fors-
chungsgemeinschaft to W. K. Peitsch (project PE-896/1).
Live cell microscopy Live cell microscopy was carried out on
A431 cells stably transfected with drebrin–EGFP–C1 and –N1 con-
structs, grown on plastic dishes in small colonies for 1 d. Time
lapse images were taken at intervals of 30 s for a maximal period of
30 min, using a Zeiss Axiovert S100 TV microscope with an Open-
lab 3.1.7 alias software.
Supplementary Material
The following material is available online for this article.
Table S1 Distribution of drebin-EGFP in stably transfected human
vulvar epidermoid carcinoma cells of line A431, compared to other
proteins.

1
25 : 4 OCTOBER 2005
DREBRIN IN EPITHELIAL SKIN CANCERS 773
DOI: 10.1111/j.0022-202X.2005.23793.x
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