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 (^{Peitsch et al, 1999,2003}). But not much was known about drebrin in stratified squamous epithelia, including the epidermis, and tumors derived therefrom. Therefore, examination 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 reactions 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 parallel 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).

Immunostaining on cryostat and paraffin-embedded sections of normal human skin (Figure 1) showed that the epidermis was essentially negative for drebrin (Figure 1a). Drebrin was, however, enriched in the ducts of eccrine sweat glands, especially along their cell–cell boundaries (Figure 1b), whereas sebaceous glands did not display detectable amounts. In addition, intercellular junctions of hair follicles were stained, both in the bulbs (Figure 1c) and, in upper parts, in a layer apparently corresponding to the outer root sheath (ORS; Figure 1d). As previously reported (^{Peitsch et al, 1999}), small blood vessels in the dermis were also drebrin-positive (asterisks in Figures 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.

Figure 1.

Immunolocalization of drebrin in paraffin-embedded sections of normal human skin. Normal human epidermis (bracket in A) contains almost no drebrin, but immunoreactions are seen in the endothelium of small blood vessels (asterisks 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 micrographs are shown in A'–D'. A–D, antiserum dreb4.1gp. Scale bars: 25 m.

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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, drebrin labeling was somewhat weaker (Figures 2a –a", transverse section; Figure 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 protein 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 (Figure 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 accumulated along cell boundaries, whereas cytokeratin 14 was positive throughout the cytoplasm (Figure 2d).

Figure 2.

Double-label confocal immunofluorescence microscopy of human hair follicles. Formaldehyde-fixed cryostat sections of hair follicles were stained for drebrin with monoclonal antibody Mx823 (red in A, A", and B–D), in combination with antisera against different hair keratins (green). Both drebrin and hair keratin 6hf (green in A', A", and B) are enriched in the companion layer (A–A", vertical section; B, longitudinal section). In addition, drebrin antibodies react in the outer sheath (ORS) (A, A", and B–D) whereas the Huxley layer of the inner root sheath, labeled by antibodies against hair keratin 6irs4 (green in C), does not present significant amounts of drebrin (C, longitudinal section). In D, co-existence of cytokeratin 14 (green) and drebrin in the ORS is shown. Asterisks in A and A" denote drebrin-positive eccrine sweat gland ducts. Scale bars: 100 m.

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Immunolocalization of drebrin in epithelial skin tumors

As the gene expression patterns of several junction-associated 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 examined, using drebrin-specific antibodies (Table I). In BCC, intense drebrin reactivity was observed in all specimens examined (Figure 3a). This conspicuously positive reaction was seen throughout the entire tumor on formaldehyde-fixed cryostat sections and paraffin-embedded tissues. On cryostat 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).

Figure 3.

Drebrin distribution in different epithelial skin tumors. Immunohistochemical 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 dreb4.1gp; A'–D', phase-contrast images. Scale bars: 25 m.

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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 appeared to be negative for drebrin (Figure 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 differentiation. 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 intensely drebrin-positive and drebrin-negative tumor regions, was observed in keratoacanthomas (Figures 3c,d). Precancerous lesions such as actinic keratoses showed similar heterogeneity. Here, in some regions, the basal keratinocytes revealed some cell border drebrin staining, whereas other basal regions and the upper epidermal layers remained drebrin-negative (Table I).

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 practically 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 certain 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 sebaceous glands, eccrine sweat glands, and hair follicles, and also at cell borders in endothelia of small blood vessels (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, homogeneously 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 (Figures 4a –a"). Double immunostaining with different marker proteins of adhering junction plaques such as -catenin, -catenin, and plakoglobin showed a far-reaching overlap with drebrin (data not shown). In contrast, the desmosomal plaque protein, desmoplakin, was completely differently distributed, showing the typical punctuate desmosomal arrays whereas drebrin immunostaining displayed a more linear and, in some regions, distinctive interdesmosomal pattern (Figures 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 claudin-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-localization 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.

Figure 4.

Double-label immunofluorescence confocal microscopy, showing drebrin accumulation at adhering junctions of basal cell carcinomas (BCC). When formaldehyde-fixed cryostat sections of BCC have been double labeled for drebrin (mab Mx823, red in A and A') and actin (phalloidin Alexa 488, green in A and A"), far-reaching co-localization of both proteins in the submembranous actin cortex underlying adhering junctions is seen (A, merge). In contrast, drebrin and desmoplakin antibodies (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 m.

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As our double label laser scanning results indicated accumulation of drebrin near adhering junctions, we also performed immunoelectron microscopy (Figures 5b –e). On sections of BCC, immunogold labeling for drebrin was detected in the microfilament network underlying the plaques of adhering junctions, usually close to the plaques, whereas desmosomes were not significantly labeled. For comparison, immunoelectron microscopy with antibodies to -catenin was performed, which showed a typical junctional plaque reaction, typically closer to the plasma membrane than drebrin (data not shown).

Figure 5.

Electron and immunoelectron microscopy of basal cell carcinomas (BCC). The conventional electron micrograph presented in A shows two neighboring BCC, connected by a region rich in junctions, i.e., desmosomes (arrows) and punctum-like adhering junctions (asterisks), the latter usually characterized by a smaller junctional plaque. (B–E) When cryostat sections of BCC have been reacted for ultrathin section electron microscopy with drebrin antibodies (B–E, monoclonal antibody Mx823), in combination with secondary gold-conjugated immunoglobulins and silver enhancement, prominent reactions are seen along intercellular adhering junctions (B, C), sometimes close to, but distinctly separate from desmosomal plaques (D–E, higher magnifications). N, nucleus. Scale bars: 500 nm (A, B); 200 nm (C–E).

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Immunoblot analysis of skin and skin tumors

To compare the amounts of drebrin in the skin and in skin tumors, we performed immunoblot analysis of total protein lysates from normal skin (Figures 6a –a ",lane 1), scalp (lane 2), BCC (lane 3), squamous carcinomas (lane 4), melanoma metastases (lane 5), and leiomyosarcoma (lane 6). An immunoreactive 120 kDa band corresponding to drebrin was hardly detectable in normal skin and scalp but was drastically increased in all tumors examined. Comparably high amounts of drebrin were found in lysates of BCC, corresponding to our immunostaining results, and in SCC, despite the microscopically observed heterogeneous staining pattern. In contrast to drebrin, the amounts of actin were approximately equal in all samples examined, with the expected exception of the actin-rich leiomyosarcoma (Figure 6a").

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 leiomyosarcoma (lane 6), have been loaded in approximately equal amounts and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (A, Coomassie Brilliant Blue-stained gel). Immunoblot analysis, using antibodies to drebrin (A'), shows a very faint immunoreactive 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 interfollicular 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.

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Primary human keratinocytes synthesize drebrin

When primary human keratinocytes were analyzed by immunofluorescence microscopy and immunoblotting (Figure 7), drebrin could be detected in rather high amounts. Drebrin and actin again co-localized along cell–cell boundaries, corresponding to our observations on BCC (Figure 7a). Moreover, in subconfluent 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 (Figure 7b, lane 1) and confluent (lane 2) keratinocytes showed strong drebrin reactions. Thus, the cell culture conditions appeared to have induced drebrin synthesis in normally near drebrin-negative keratinocytes.

Figure 7.

Induction and localization of drebrin in primary cultured keratinocytes. When human keratinocytes cultured for two passages have been stained with drebrin antibodies (red in A and A', monoclonal antibody Mx823), in combination with phalloidin to visualize actin filaments (green in A and A"), confocal laser scanning microscopy shows accumulation of drebrin along cell–cell borders, suggestive of a localization at intercellular adherent junctions (A'), in far-reaching co-localization with phalloidin (A"). By contrast, the cytoplasmic actin stress fibers do not contain drebrin (A, merge). Scale bar: 10 m. (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 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.

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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 elucidate such functions, we generated cell lines stably expressing drebrin–EGFP fusion proteins. We chose A431 cells as this was one of the rare cell lines devoid of endogenous drebrin (Figure 8a', lane 2; see also^{Peitsch et al, 1999,2001}). Such cells were transfected—transiently and stably—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 numerous actin-rich cell processes (data not shown; for relevant references, see Introduction). In the transfectants seemingly containing less drebrin, however, the fusion protein was enriched at cell–cell boundaries, and fewer and shorter cell processes were seen (Figure 9, left panel). For further experiments, stable lines exhibiting the latter phenotype were analyzed. Three such lines (Figure 8a', lanes 3–5) 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.

Figure 8.

Immunoblot analysis and immunoprecipitation of drebrin-containing proteins in A431 cells stably transfected with drebrin-enhanced 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 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 immunoprecipitation control (lane 2), the only prominent band is seen at approximately 55 kDa, containing the heavy chains of the immunoglobulins (see also lanes 4 and 6). The proteins bound non-specifically during "preclearing" 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).

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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 antibodies to different junctional proteins (red 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 intercellular borders, although some diffuse cytoplasmic staining is also seen. Additionally, some cells form numerous small cell processes, extending to different focal planes (asterisks in A, A", D, and D"). When simultaneous staining is performed with antibodies to the adhering junction-associated proteins plakoglobin (A'), -catenin (B'), and E-cadherin (C'), the merge images (A"–C") show partial co-localization with drebrin–EGFP at cell–cell borders, more prominently for -catenin and E-cadherin than for plakoglobin. Vinculin antibodies react with focal contacts (D'), that do not contain drebrin (D", merge). Scale bars: 20 m.

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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 (Figure 8b). In immunoprecipitates of both drebrin–EGFP–C1 transfectants (lane 4) and of 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 excised 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 (Figure 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).

When A431 cells stably transfected with drebrin–EGFP constructs were immunostained with antibodies and compared with constituent proteins of intercellular junctions, far-reaching co-localization was evident (Figure 9);(Table S1). Antibodies against plakoglobin (Figures 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 desmosomal structures were drebrin negative. Proteins representing exclusive "markers" for adhering junctions such as the plaque proteins -catenin (Figures 9b 'b "), -catenin, and p120 (not shown) as well as the transmembrane protein E-cadherin (Figures 9c 'c ") partially co-localized with drebrin (see merged images in Figures 9b 'b "). When cells were 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 desmoplakin showed punctate staining patterns entirely different from those of the drebrin fusion proteins (not shown). No significant co-localization was seen with protein ZO-1, 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). Antibodies against -actinin (not shown), vinculin (Figures 9d 'd "), and vasodilator-stimulated phosphoprotein (VASP) (not shown) reacted predominantly with focal adhesions that were virtually devoid of drebrin–EGFP, as previously reported 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 L EGF and analyzed by live cell microscopy, rapid formation of lamellipodia was observed within a few minutes (Figures 10b,c; Supplementary Material, Movie 1). Within the next minutes, the lamellipodia retracted (Figures 10d,e), and filopodia-like structures appeared (Figures 10f –h). During this process, the drebrin–EGFP fusion proteins remained for the most part enriched along cell boundaries (Figures 10a'–H'; Supplementary Material, Movie 2), and after retraction even showed enhanced fluorescence at these sites (arrows in Figures 10g ' h'). In some cells, drebrin–EGFP was, in addition, temporarily enriched in spikes in extended (asterisks in Figures 10b ' c ') and retracting lamellipodia (asterisks in Figures 10d ' f'). This signal, however, was rather faint compared with the junctional signal, and the filopodia formed after approximately 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 L (not shown), and with EGFP constructs at the N- and the C-terminus of drebrin.

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 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 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 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 m.

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In summary, our observations that drebrin is not accumulated in the filopodia of subconfluent keratinocytes and the live cell microscopy results all indicate that in the epithelial cells examined, drebrin normally functions at junctions, not in filopodia and lamellipodia. Overexpression of drebrin, however, may lead to the formation of cell processes, as previously described for other cells (e.g.,^{Shirao et al, 1992;Hayashi and Shirao, 1999;Keon et al, 2000}).

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, 1999,2003;Keon et al, 2000}). Moreover, drebrin is accumulated in the ORS and the companion layer of hair follicles, most likely in association with intercellular adhering junctions (zonulae adhaerentes). This distribution pattern appears rather unexpected, 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, -catenin (^{Gat et al, 1998;Celso et al, 2004}) and other plaque proteins of adhering junctions such as -catenin and plakoglobin have been shown to control hair follicle morphogenesis and cycling (^{Charpentier et al, 2000;Vasioukhin 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 morphogenesis, we will study hair follicle development, murine hair follicle 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 et al, 1999;Krüger 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 conspicuous by its drebrin content. A follicular derivation of BCC might explain why BCC are homogeneously drebrin positive, 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 overlooked and of small nests at the margins of surgical resections. Moreover, it will also have to be examined as to whether high amounts of drebrin occur in other tumors derived 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 mosaicism 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 indicate that epithelial skin cancers can comprise multiple small subregions and that such regionalization might contribute to notorious differences of chemoresistency of several 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 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 commonly related to tumorigenesis, which will have to be systematically examined in future studies. In this context, our finding that drebrin is virtually undetectable in biopsy specimens 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, -catenin is widely regarded as a regulator of tumorigenesis (review:^{Nelson and Nusse, 2004}; see also^{Chan et al, 1999;Doglioni et al, 2003;El-Bahrawy et al, 2003}).

Drebrin localization and morphogenic effects in epithelial 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 higher content of drebrin (Figure 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 - and -catenin and to anchor the actin microfilament bundle system (see, e.g.,^{Green et al, 1987;Kaiser et al, 1993;Kübler 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 adhering 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 types (^{Shirao et al, 1992;Hayashi and Shirao, 1999;Toda et al, 1999}).

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 attachment 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 observations and with reports from the literature, the results shown here suggest that drebrin can serve diverse important 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 migratory and adhesive activities of tumor cells.

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 (Biochrom, Berlin, Germany) plus 2 mM glutamine, and were subdivided twice weekly at a 1:10 ratio. For stimulation with EGF (Sigma, Deisenhofen, Germany), cells were fed with serum-free medium and, after 2 d, EGF was added in concentrations between 10 and 100 ng per mL. Stimulated cells and non-stimulated controls were further analyzed 0–25 min after stimulation. Primary human keratinocytes were grown using Keratinocyte Growth Medium 2 Kit (PromoCell, Heidelberg, Germany) at normal calcium concentration (1.5 mM CaCl₂). Cells were subcultured once per week 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 described studies. The study was conducted according to the Declaration of Helsinki Principles.

Cloning of drebrin–EGFP constructs and myc-tagged constructs

The full-length cDNA encoding drebrin E2 (IMAGE ID: 3356428) was obtained from Resource Center/Primary Database (RZPD, clone ID: IMAGp958A05162, in a pOTB7 vector). For cloning into vector pEGFP–C1 (BD Biosciences Clontech, Palo Alto, California), a Bgl II restriction site was generated by PCR at the N-terminus of the drebrin E2 sequence (forward primer: 5'-TTT AGA 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 I and a Bam HI site (forward primer: 5'-GGT ACT TCA GTC AAT CAC AGG-3'; reverse primer: 5'-TTT CTC GAG GAT CCT CAC CAC 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 the drebrin–pOTB vector (forward primer: 5'-AAG AAT TCA ATG GCC GGC GTC AGC TTC AGC-3'; reverse primer: 5'-CGC ACT TGC GGG CAT CAG GCA CAT-3'). The drebrin sequence was subsequently cloned into the pcDNA3.1 vector containing the myc-tag with Eco RI and Xho I restriction. All PCR products were verified by DNA sequencing.

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-positive 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 nitrogen at approximately -130°C and stored at -80°C. For other experiments, aldehyde-fixed, paraffin-embedded tissues were used. Procedures were performed with patients' informed consent.

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 487–502, STLQGEPRAPTPPSGT), and pep4 (aa 632–649, 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 g of the peptide cocktail diluted in phosphate-buffered saline (PBS) to a volume of 150 L, supplemented with 150 L of complete Freud's adjuvants (Sigma). Three subcutaneous booster injections with the same amount of peptide cocktail mixed with 150 L incomplete Freud's adjuvant were given at intervals of 21 d. At day 63, a final boost with 30 g of the peptide cocktail diluted in 200 L PBS was injected intraperitoneally. Four days thereafter, the mice were sacrificed, spleen cells were harvested and fused with mouse myeloma cells of line SP2/0 at a ratio of 2:1 in the presence of polyethylenglycol 1500 (Roche Diagnostics, Mannheim, Germany). After fusion, the cells were placed on 24-well microtiter plates and cultured in hypoxanthine–aminopterin–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.^{Peitsch 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, hybridomas were weaned off HAT–RMPI into RMPI medium alone and grown in 25 cm² flasks or propagated as ascites in Pristane-treated BALB/c mice. Immunoglobulin subclasses were determined and one of the drebrin-specific immunoglobulin G (IgG) mab obtained, Mx823, was further characterized by separate preincubation with the peptides used for immunization at concentrations ranging from 10 to 200 g per mL in PBS, followed by immunofluorescence microscopy and immunoblot analysis. These experiments 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).

Pep4 was also used for generation of antibodies in guinea-pigs (cf.^{Peitsch et al, 2001}). The resulting antiserum dreb4.1gp also 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 (^{Peitsch et al, 2001,2003}).

Other antibodies and reagents

For comparison, we also used the murine drebrin mab M2F6 against purified chicken drebrin E, purchased from MoBiTec (Göttingen, Germany). Further mab applied in this study were against plakoglobin (clone PG5.1, Progen Biotechnik, and clone 11E4, kindly provided by Dr M. J. Wheelock, University of Nebraska Medical Center, Omaha, Nebraska), desmogleins Dsg 1 and 2 (clone DG3.10; Progen Biotechnik), E-cadherin, proteins p120^ctn, VASP, and Mena (all from BD Biosciences Pharmingen, Heidelberg, Germany), vinculin (clones 11.5 and hvinc-1), ezrin and -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 -catenin, -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 Laboratories (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 6irs4 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 microfilaments were stained with phalloidin coupled to Alexa 488 (MoBiTec) or TRITC-labeled phalloidin (Sigma).

For immunofluorescence microscopy, primary antibody complexes were visualized with secondary antibodies coupled to Cy-3 (Dianova) or Alexa 488 (MoBiTec). For immunoblot analysis, horseradish peroxidase-conjugated secondary antibodies were applied in combination with the enhanced chemiluminescence system (NEN, Köln, Germany).

Immunofluorescence and confocal laser scanning microscopy

Cultured cells grown on glass coverslips were fixed in 2% formaldehyde for 20 min at room temperature (RT) and treated with 50 mM NH₄Cl 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 (-20°C, 5 min) and acetone (-20°C, 20 s). Frozen tissues were sectioned at 4–5 m thickness, using a Leica cryomicrotome (Bensheim, Germany), air-dried and fixed with 2% formaldehyde or, alternatively, with acetone (-20°C, 10 min). Prior to immunostaining, sections were blocked with 5% goat serum for 20 min. For sectioning of paraffin-embedded tissues (3–4 m), a Jung Histoslide 2000R cryomicrotome (Leica) was used. After deparaffinization according to standard techniques, sections were pre-treated by microwaving in 100 mM TRIS buffer containing 5% urea (pH 9.5, 10 min, 98°C) to achieve heat-induced antigen retrieval. Sections were then washed with PBS (5 min, RT), incubated with PBS containing 2% milk powder (Carl Roth, Karlsruhe, Germany) 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 performed as described (^{Peitsch et al, 2001}), using an Axiophote II photomicroscope (Carl Zeiss, Jena, Germany) for immunofluorescence microscopy and a Zeiss LSM 510 UV microscope for confocal microscopy.

Live cell microscopy

Live cell microscopy was carried out on A431 cells stably transfected with drebrin–EGFP–C1 and –N1 constructs, 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 Openlab 3.1.7 alias software.

Electron and immunoelectron microscopy

Conventional electron microscopic analysis of samples of BCC was performed according to the protocol described in^{Peitsch et al (2003)}. For immunoelectron microscopy, cryostat sections of BCC of 4–5 m thickness were fixed with 2% formaldehyde for 10 min (RT), followed by treatment with 50 mM NH₄Cl (5 min) and permeabilization with 0.1% saponin (5 min). Staining and embedding were as previously reported (^{Peitsch et al, 2003}). Primary antibodies were Mx823 (1:50) and the rabbit antiserum to -catenin, secondary antibodies anti-mouse, or anti-rabbit immunoglobulins conjugated with 1.4 nm-gold particles (Nanogold; Biotrend, Köln, Germany). Silver enhancement was performed for 5 min with a silver enhancement 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.

Immunoblotting, immunoprecipitation, and MALDI analysis

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 precooled 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 (4°C), 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 (4°C). 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 buffer (^{Peitsch et al, 1999}), boiled for 3 min at 95°C, 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. Schnölzer (Protein Analysis Facility, German Cancer Research Center), as described (^{Peitsch et al, 1999}).

References

1. Behrens, J, Mareel, MM, Van Roy, FM, Birchmeier, W: Dissecting tumor cell invasion: Epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cel–cell adhesion. J Cell Biol 1989 108:2435–2447,  | Article | PubMed | ISI | ChemPort |
2. Bretscher, A, Edwards, K, Fehon, RG: ERM proteins and merlin: Integrators at the cell cortex. Nat Rev Mol Cell Biol 2002 3:586–599,  | Article | PubMed | ISI | ChemPort |
3. Butkevich, E, Hulsmann, S, Wenzel, D, Shirao, T, Duden, R, Majoul, I: Drebrin is a novel connexin-43 binding partner that links gap junctions to the submembrane cytoskeleton. Curr Biol 2004 14:650–658,
4. Cavallaro, U, Christofori, G: Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nat Rev Cancer 2004 4:118–132,  | Article | PubMed | ISI | ChemPort |
5. Celso, CL, Prowse, DM, Watt, FM: Transient activation of beta-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours. Development 2004 131:1787–1799,  | Article | PubMed | ISI | ChemPort |
6. Chan, EF, Gat, U, McNiff, JM, Fuchs, E: A common human skin tumour is caused by activating mutations in beta-catenin. Nat Genet 1999 21:410–413,  | Article | PubMed | ISI | ChemPort |
7. Charpentier, E, Lavker, RM, Acquista, E, Cowin, P: Plakoglobin suppresses epithelial proliferation and hair growth in vivo. J Cell Biol 2000 149:503–520,  | Article | PubMed | ISI | ChemPort |
8. Cohen, MM, Jr: The hedgehog signalling network. Am J Med Genet 2003 123A:5–28,
9. Cowin, P, Kapprell, HP, Franke, WW, Tamkun, J, Hynes, RO: Plakoglobin: A protein common to different kinds of intercellular adhering junctions. Cell 1986 46:1063–1073,  | Article | PubMed | ISI | ChemPort |
10. Cribier, B, Peltre, B, Langbein, L, Winter, H, Schweizer, J, Grosshans, E: Expression of type I hair keratins in follicular tumours. Br J Dermatol 2001 144:977–982,  | Article | PubMed |
11. DeMali, KA, Burridge, K: Coupling membrane protrusion and cell adhesion. J Cell Sci 2003 116:2389–2397,  | Article | PubMed | ISI | ChemPort |
12. Doglioni, C, Piccinin, S, Demontis, S, et al: Alterations of beta-catenin pathway in non-melanoma skin tumors: Loss of alpha-ABC nuclear reactivity correlates with the presence of beta-catenin gene mutation. Am J Pathol 2003 163:2277–2287,  | PubMed | ChemPort |
13. El-Bahrawy, M, El-Masry, N, Alison, M, Poulsom, R, Fallowfield, M: Expression of beta-catenin in basal cell carcinoma. Br J Dermatol 2003 148:964–970,  | Article | PubMed | ChemPort |
14. Franke, WW, Moll, R, Mueller, H, et al: Immunocytochemical identification of epithelium-derived human tumors with antibodies to desmosomal plaque proteins. Proc Natl Acad Sci USA 1983 80:543–547,  | Article | PubMed | ChemPort |
15. Garrod, DR: Desmosomes and cancer. Cancer Surv 1995 24:97–111,  | PubMed |
16. Gat, U, DasGupta, R, Degenstein, L, Fuchs, E: De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin. Cell 1998 95:605–614,  | Article | PubMed | ISI | ChemPort |
17. Green, KJ, Geiger, B, Jones, JC, Talian, JC, Goldman, RD: The relationship between intermediate filaments and microfilaments before and during the formation of desmosomes and adherens-type junctions in mouse epidermal keratinocytes. J Cell Biol 1987 104:1389–1402,  | Article | PubMed | ISI | ChemPort |
18. Haftek, M, Hansen, MU, Kaiser, HW, Kreysel, HW, Schmitt, D: Interkeratinocyte adherens junctions: Immunocytochemical visualization of cell–cell junctional structures, distinct from desmosomes, in human epidermis. J Invest Dermatol 1996 106:498–504,  | Article | PubMed | ISI | ChemPort |
19. Hahn, H, Wicking, C, Zaphiropoulous, PG, et al: Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 1996 85:841–851,  | Article | PubMed | ISI | ChemPort |
20. Hayashi, K, Ishikawa, R, Kawai-Hirai, R, Takagi, T, Taketomi, A, Shirao, T: Domain analysis of the actin-binding and actin-remodeling activities of drebrin. Exp Cell Res 1999 253:673–680,
21. Hayashi, K, Shirao, T: Change in the shape of dendritic spines caused by overexpression of drebrin in cultured cortical neurons. J Neurosci 1999 19:3918–3925,  | PubMed | ISI | ChemPort |
22. Heid, HW, Schmidt, A, Zimbelmann, R, et al: Cell type-specific desmosomal plaque proteins of the plakoglobin family: Plakophilin 1 (band 6 protein). Differentiation 1994 58:113–131,  | Article | PubMed | ISI | ChemPort |
23. Hirohashi, S, Kanai, Y: Cell adhesion system and human cancer morphogenesis. Cancer Sci 2003 94:575–581,  | Article | PubMed | ISI | ChemPort |
24. Ikeda, K, Shirao, T, Toda, M, Asada, H, Toya, S, Uyemura, K: Effect of a neuron-specific actin-binding protein, drebrin A, on cell–substratum adhesion. Neurosci Lett 1995 194:197–200,
25. Jamora, C, Fuchs, E: Intercellular adhesion, signalling and the cytoskeleton. Nat Cell Biol 2002 4:E101–E108,  | Article | PubMed | ISI | ChemPort |
26. Jih, DM, Lyle, S, Elenitsas, R, Elder, DE, Cotsarelis, G: Cytokeratin 15 expression in trichoepitheliomas and a subset of basal cell carcinomas suggests they originate from hair follicle stem cells. J Cutan Pathol 1999 26:113–118,  | Article | PubMed | ISI | ChemPort |
27. Jones, JC, Grelling, KA: Distribution of desmoplakin in normal cultured human keratinocytes and in basal cell carcinoma cells. Cell Motil Cytoskeleton 1989 13:181–194,
28. Kaiser, HW, Ness, W, Jungblut, I, Briggaman, RA, Kreysel, HW, O'Keefe, EJ: Adherens junctions: Demonstration in human epidermis. J Invest Dermatol 1993 100:180–185,  | Article | PubMed | ISI | ChemPort |
29. Keon, BH, Jedrzejewski, PT, Paul, DL, Goodenough, DA: Isoform specific expression of the neuronal F-actin binding protein, drebrin, in specialized cells of stomach and kidney epithelia. J Cell Sci 2000 113:325–336,
30. Kovacs, EM, Goodwin, M, Ali, RG, Paterson, AD, Yap, AS: Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts. Curr Biol 2002 12:379–382,  | Article | PubMed | ISI | ChemPort |
31. Krüger, K, Blume-Peytavi, U, Orfanos, CE: Basal cell carcinoma possibly originates from the outer root sheath and/or the bulge region of the vellus hair follicle. Arch Dermatol Res 1999 291:253–259,  | Article | PubMed | ISI | ChemPort |
32. Krunic, AL, Garrod, DR, Smith, NP, Orchard, GS, Cvijetic, OB: Differential expression of desmosomal glycoproteins in keratoacanthoma and squamous cell carcinoma of the skin: An immunohistochemical aid to diagnosis. Acta Derm Venereol 1996 76:394–398,  | PubMed | ISI | ChemPort |
33. Kübler, MD, Watt, FM: Changes in the distribution of actin-associated proteins during epidermal wound healing. J Invest Dermatol 1993 100:785–789,  | Article | PubMed | ISI | ChemPort |
34. Kurzen, H, Esposito, L, Langbein, L, Hartschuh, W: Cytokeratins as markers of follicular differentiation: An immunohistochemical study of trichoblastoma and basal cell carcinoma. Am J Dermatopathol 2001 23:501–509,
35. Kurzen, H, Munzing, I, Hartschuh, W: Expression of desmosomal proteins in squamous cell carcinomas of the skin. J Cutan Pathol 2003 30:621–630,  | Article | PubMed | ISI |
36. Langbein, L, Pape, UF, Grund, C, et al: Tight junction-related structures in the absence of a lumen: Occludin, claudins and tight junction plaque proteins in densely packed cell formations of stratified epithelia and squamous cell carcinomas. Eur J Cell Biol 2003a 82:385–400,  | Article | PubMed | ISI | ChemPort |
37. Langbein, L, Rogers, MA, Praetzel, S, Aoki, N, Winter, H, Schweizer, J: A novel epithelial keratin, hK6irs1, is expressed differentially in all layers of the inner root sheath, including specialized huxley cells (Flugelzellen) of the human hair follicle. J Invest Dermatol 2002 118:789–799,  | Article | PubMed | ISI | ChemPort |
38. Langbein, L, Rogers, MA, Praetzel, S, Winter, H, Schweizer, J: K6irs1, K6irs2, K6irs3, and K6irs4 represent the inner-root-sheath-specific type II epithelial keratins of the human hair follicle. J Invest Dermatol 2003b 120:512–522,  | Article | PubMed | ISI | ChemPort |
39. Llorens, A, Rodrigo, I, Lopez-Barcons, L, et al: Down-regulation of E-cadherin in mouse skin carcinoma cells enhances a migratory and invasive phenotype linked to matrix metalloproteinase-9 gelatinasen expression. Lab Invest 1998 78:1131–1142,  | PubMed | ISI | ChemPort |
40. Lozano, E, Cano, A: Induction of mutual stabilization and retardation of tumor growth by coexpression of plakoglobin and E-cadherin in mouse skin spindle carcinoma cells. Mol Carcinog 1998 21:273–287,  | Article | PubMed | ISI |
41. Malliri, A, Symons, M, Hennigan, RF, et al: The transcription factor AP-1 is required for EGF-induced activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells. J Cell Biol 1998 143:1087–1099,  | Article | PubMed | ISI | ChemPort |
42. Mammoto, A, Sasaki, T, Asakura, T, et al: Interactions of drebrin and gephyrin with profilin. Biochem Biophys Res Commun 1998 243:86–89,  | Article | PubMed | ISI | ChemPort |
43. Mareel, M, Vleminckx, K, Vermeulen, S, Yan, G, Bracke, M, van Roy, F: Downregulation in vivo of the invasion-suppressor molecule E-cadherin in experimental and clinical cancer. Princess Takamatsu Symp 1994 24:63–80,
44. McKoy, G, Protonotarios, N, Crosby, A, et al: Identification of a deletion in plakoglobin in arrhythmogenic right ventricular cardiomyopathy with palmoplantar keratoderma and woolly hair (Naxos disease). Lancet 2000 355:2119–2124,  | Article | PubMed | ISI | ChemPort |
45. Mertens, C, Kuhn, C, Moll, R, Schwetlick, I, Franke, WW: Desmosomal plakophilin 2 as a differentiation marker in normal and malignant tissues. Differentiation 1999 64:277–290,
46. Moll, I, Kurzen, H, Langbein, L, Franke, WW: The distribution of the desmosomal protein, plakophilin 1, in human skin and skin tumors. J Invest Dermatol 1997 108:139–146,  | Article | PubMed | ISI | ChemPort |
47. Moll, R, Cowin, P, Kapprell, HP, Franke, WW: Desmosomal proteins: New markers for identification and classification of tumors. Lab Invest 1986 54:4–25,  | PubMed | ISI | ChemPort |
48. Moll, R, Mitze, M, Frixen, UH, Birchmeier, W: Differential loss of E-cadherin expression in infiltrating ductal and lobular breast carcinomas. Am J Pathol 1993 143:1731–1742,  | PubMed | ISI | ChemPort |
49. Nakanishi, Y, Ochiai, A, Akimoto, S, et al: Expression of E-cadherin, alpha-catenin, beta-catenin and plakoglobin in esophageal carcinomas and its prognostic significance: Immunohistochemical analysis of 96 lesions. Oncology 1997 54:158–165,  | PubMed | ISI | ChemPort |
50. Nelson, WJ, Nusse, R: Convergence of Wnt, beta-catenin, and cadherin pathways. Science 2004 303:1483–1487,  | Article | PubMed | ISI | ChemPort |
51. Nuber, UA, Schäfer, S, Schmidt, A, Koch, PJ, Franke, WW: The widespread human desmocollin Dsc2 and tissue-specific patterns of synthesis of various desmocollin subtypes. Eur J Cell Biol 1995 66:69–74,  | PubMed | ISI | ChemPort |
52. Papadavid, E, Pignatelli, M, Zakynthinos, S, Krausz, T, Chu, AC: Abnormal immunoreactivity of the E-cadherin/catenin (alpha-, beta-, and gamma-) complex in premalignant and malignant non-melanocytic skin tumours. J Pathol 2002 196:154–162,  | Article | PubMed | ISI | ChemPort |
53. Parrish, EP, Garrod, DR, Mattey, DL, Hand, L, Steart, PV, Weller, RO: Mouse antisera specific for desmosomal adhesion molecules of suprabasal skin cells, meninges, and meningioma. Proc Natl Acad Sci USA 1986 83:2657–2661,  | Article | PubMed | ChemPort |
54. Peitsch, WK, Grund, C, Kuhn, C: Drebrin is a widespread actin-associating protein enriched at junctional plaques, defining a specific microfilament anchorage system in polar epithelial cells. Eur J Cell Biol 1999 78:767–778,  | PubMed | ISI | ChemPort |
55. Peitsch, WK, Hofmann, I, Endlich, N, et al: Cell biological and biochemical characterization of drebrin complexes in mesangial cells and podocytes of renal glomeruli. J Am Soc Nephrol 2003 14:1452–1463,
56. Peitsch, WK, Hofmann, I, Prätzel, S, et al: Drebrin particles: Components in the ensemble of proteins regulating actin dynamics of lamellipodia and filopodia. Eur J Cell Biol 2001 80:567–579,  | Article | PubMed | ISI | ChemPort |
57. Perl, AK, Wilgenbus, P, Dahl, U, Semb, H, Christofori, G: A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature 1998 392:190–193,  | Article | PubMed | ISI | ChemPort |
58. Schäfer, S, Stumpp, S, Franke, WW: Immunological identification and characterization of the desmosomal cadherin Dsg2 in coupled and uncoupled epithelial cells and in human tissues. Differentiation 1996 60:99–108,  | Article | PubMed | ISI | ChemPort |
59. Schirren, CG, Rutten, A, Kaudewitz, P, Diaz, C, McClain, S, Burgdorf, WH: Trichoblastoma and basal cell carcinoma are neoplasms with follicular differentiation sharing the same profile of cytokeratin intermediate filaments. Am J Dermatopathol 1997 19:341–350,  | Article | PubMed | ISI | ChemPort |
60. Shimoyama, Y, Hirohashi, S: Cadherin intercellular adhesion molecule in hepatocellular carcinomas: Loss of E-cadherin expression in an undifferentiated carcinoma. Cancer Lett 1991 57:131–135,  | Article | PubMed | ISI | ChemPort |
61. Shirao, T: The roles of microfilament-associated proteins, drebrins, in brain morphogenesis: A review. J Biochem (Tokyo) 1995 117:231–236,  | ChemPort |
62. Shirao, T, Kojima, N, Obata, K: Cloning of drebrin A and induction of neurite-like processes in drebrin-transfected cells. Neuroreports 1992 3:109–112,
63. Tada, J, Hashimoto, K: Ultrastructural localization of gap junction protein connexin 43 in normal human skin, basal cell carcinoma, and squamous cell carcinoma. J Cutan Pathol 1997 24:628–635,  | PubMed | ISI | ChemPort |
64. Takahashi, H, Sekino, Y, Tanaka, S, Mizui, T, Kishi, S, Shirao, T: Drebrin-dependent actin clustering in dendritic filopodia governs synaptic targeting of postsynaptic density-95 and dendritic spine morphogenesis. J Neurosci 2003 23:6586–6595,  | PubMed | ISI | ChemPort |
65. Takai, Y, Nakanishi, H: Nectin and afadin: Novel organizers of intercellular junctions. J Cell Sci 2003 116:17–27,  | Article | PubMed | ISI | ChemPort |
66. Toda, M, Shirao, T, Uyemura, K: Suppression of an actin-binding protein, drebrin, by antisense transfection attenuates neurite outgrowth in neuroblastoma B104 cells. Brain Res Dev Brain Res 1999 114:193–200,
67. Tsukita, S, Tsukita, S, Nagafuchi, A, Yonemura, S: Possible involvement of adherens junction plaque proteins in tumorigenesis and metastasis. Princess Takamatsu Symp 1994 24:38–50,  | PubMed |
68. Vasioukhin, V, Bauer, C, Degenstein, L, Wise, B, Fuchs, E: Hyperproliferation and defects in epithelial polarity upon conditional ablation of alpha-catenin in skin. Cell 2001 104:605–617, 119:207–235, 2003 | Article | PubMed | ISI | ChemPort |
69. Winter, H, Langbein, L, Praetzel, S, et al: A novel human type II cytokeratin, K6hf, specifically expressed in the companion layer of the hair follicle. J Invest Dermatol 1998 111:955–962,  | Article | PubMed | ISI | ChemPort |
70. Wu, H, Lotan, R, Menter, D, Lippman, SM, Xu, XC: Expression of E-cadherin is associated with squamous differentiation in squamous cell carcinomas. Anticancer Res 2000 20:1385–1390,  | PubMed | ISI | ChemPort |

Acknowledgments

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 Center) for advice on live cell microscopy. Dr Martina Schnölzer (Central Protein Analysis, German Cancer Research Center) kindly performed MALDI analyses. Silke Prätzel (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 (Department of Dermatology, Medical Center Mannheim) for keratinocyte cell culture. In addition, we would like to thank Dr Jochen Utikal (Department of Dermatology, Medical Center Mannheim) for help with preservation of tumor material and Dr Romy Porstmann (Department of Pathology, Medical Center Mannheim) for aid in pathological diagnostics. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to W. K. Peitsch (project PE-896/1).